Lessons From Models of Impaired Insulin Secretion

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285:669-684, 2003. doi:10.1152/ajpendo.00196.2003Am J Physiol Endocrinol MetabAnjaneyulu Kowluru

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invited review

Regulatory roles for small G proteins in the pancreatic

-cell: lessons from models of impaired insulin secretion

Anjaneyulu Kowluru

Department of Pharmaceutical Sciences, Applebaum College of Pharmacy and Health Sciences and the -CellBiochemistry Research Laboratory, John D. Dingell Veterans Affairs Medical Center, Detroit, Michigan 48201

Kowluru, Anjaneyulu. Regulatory roles for small G proteins in thepancreatic -cell: lessons from models of impaired insulin secretion. Am

J Physiol Endocrinol Metab 285: E669E684, 2003;10.1152/ajpendo.00196.2003.Emerging evidence suggests that GTP-binding proteins (Gproteins) play important regulatory roles in physiological insulin secre-tion from the islet -cell. Such conclusions were drawn primarily fromexperimental data derived through the use of specific inhibitors of Gprotein function. Data from gene depletion experiments appear to fur-ther substantiate key roles for these signaling proteins in the isletmetabolism. The first part of this review will focus on findings supportingthe hypothesis that activation of specific G proteins is essential forinsulin secretion, including regulation of their function by posttransla-tional modifications at their COOH-terminal cysteines (e.g., isoprenyla-tion). The second part will overview novel, non-receptor-dependentmechanism(s) whereby glucose might activate specific G proteins viaprotein histidine phosphorylation. The third section will review findingsthat appear to link abnormalities in the expression and/or functionalactivation of these key signaling proteins to impaired insulin secretion. Itis hoped that this review will establish a basis for future research in thisarea of islet signal transduction, which presents a significant potential,not only in identifying key signaling proteins that are involved inphysiological insulin secretion, but also in examining potential abnor-malities in this signaling cascade that lead to islet dysfunction and onsetof diabetes.

cytokines; posttranslational modifications; histidine phosphorylation; di-abetes mellitus; pancreatic islet

GLUCOSE-INDUCED INSULIN SECRETION from pancreatic-cells is mediated largely via the generation of solublesecond messengers, such as cyclic nucleotides, hydro-lytic products of phospholipases (A2, C, and D), andadenine nucleotides (44, 48, 59, 60, 73, 81). However,the exact molecular and cellular mechanisms underly-ing glucose-stimulated insulin secretion remain onlypartially understood. It is widely accepted that, afterits entry into the -cell (facilitated via the glucose-transporter protein GLUT2), glucose is metabolizedwith a resultant increase in the ATP/ADP ratio. Such

an increase in the intracellular ATP results in theclosure of ATP-sensitive K channels localized on theplasma membrane, as a consequence of which mem-brane depolarization occurs. This facilitates the influxof extracellular calcium through the voltage-sensitivecalcium channels. Increase in intracellular calcium isknown to be critical for the transport of insulin-con-

taining secretory granules to the plasma membrane forfusion and release of insulin into circulation (73, 81).

GTP-BINDING PROTEINS IN THE PANCREATIC -CELL

AND THEIR REGULATION BY POSTTRANSLATIONAL

MODIFICATIONS

In addition to regulation by adenine nucleotides ofglucose-stimulated insulin secretion, earlier studies(59, 73, 81) have examined the contributory roles forguanine nucleotides (i.e., GTP) in physiological insulinsecretion. For example, using selective inhibitors of the

GTP biosynthetic pathway (e.g., mycophenolic acid),several studies have documented a permissive role forGTP in insulin secretion elicited by glucose (33, 68, 66). Although the precise mechanisms underlying the reg-ulatory role(s) of GTP remain elusive, available evi-dence indicates that they might involve activation ofone (or more) G proteins (44, 48, 85). Two major groups

Address for reprint requests and other correspondence: A.Kowluru, Dept. of Pharmaceutical Sciences, 3601, Applebaum Col-lege of Pharmacy and Health Sciences, 259 Mack Ave., Wayne StateUniv., Detroit, MI 48202 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Endocrinol Metab 285: E669E684, 2003;10.1152/ajpendo.00196.2003.

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of G proteins have been identified in -cells (44, 48, 85).The first group consists of trimeric G proteins com-prised of (3943 kDa)-, (3537 kDa)-, and (68kDa)-subunits. These are involved in the coupling of various receptors to their intracellular effectors, suchas adenylate cyclase, phosphodiesterase, or phospho-lipases (6, 17, 85). The second group of G proteins(which is the main focus of this review) is comprised of

small-molecular-mass (2025 kDa) monomeric G pro-teins, which are involved in protein sorting as well astrafficking of secretory vesicles (see Refs. 39, 44, 48 forreviews). A large body of evidence indicates that thisfamily of G proteins undergoes posttranslational mod-ifications, such as isoprenylation and carboxyl methyl-ation, at their COOH-terminal cysteine residues (oftenreferred to as the CAAX motif; 39, 44, 48).

The first of a four-step modification sequence (Fig. 1)includes incorporation of a 15-carbon (farnesyl) or 20-carbon (geranylgeranyl) isoprenoid moiety, which isderived from mevalonic acid (MVA), onto a cysteineresidue toward the carboxyl terminus of the candidateG proteins. This is followed by proteolysis of several

amino acids (up to a maximum of three). A carboxylmethylation step then modifies the newly exposed car-boxylate anion of the cysteine. In some cases, thecovalent addition of a long-chain fatty acid, typicallypalmitate, at cysteine residues, which are upstream tothe CAAX motif, completes the cascade. Such modifi-cation(s) are thought to render the modified G proteinsmore hydrophobic and enable them to associate with

membranes for interaction with their respective effec-tors (39, 44, 48, 92). Because the isoprenylation of Gproteins occurs shortly after their synthesis, and be-cause half-lives of prenylated proteins are ratherlong, this is not likely to be an acute regulatory step;however, in many cases, prenylation is necessary toallow candidate G proteins to intercalate into the rel-evant membrane compartment. In contrast, the meth-ylation and acylation steps (Fig. 1) are subject to acuteregulation at the level of the on steps (i.e., addition ofmethyl or acyl groups) as well as the off steps (i.e.,deletion of methyl or acyl groups). The addition andremoval of methyl groups are catalyzed by carboxylmethyl transferase and esterase, respectively. Like-

Fig. 1. Posttranslational modifications of small G proteins. The first of the four-step reaction is incorporation ofeither a 15 (farnesyl)- or a 20 (geranylgeranyl)-carbon derivative of mevalonic acid (MVA) into the COOH-terminalcysteine via a thioether linkage. This reaction is catalyzed by either the farnesyl or geranylgeranyl transferases,respectively. After this, the three amino acids after the prenylated cysteine are removed by a protease ofmicrosomal origin, thereby exposing the carboxylate anion. This site is then methylated by a carboxyl methyltransferase, which transfers a methyl group onto the carboxylate group using S-adenosyl methionine (SAM) as themethyl donor. We have shown that the carboxyl methylation of speci fic G proteins (e.g., Cdc42) increases theirhydrophobicity and translocation to the membrane fraction (see text for additional details). In addition to these,certain G proteins (e.g., H-Ras) have also been shown to undergo palmitoylation at a cysteine residue, which isupstream to the prenylated cysteine. It is thought that palmitoylation provides a firm anchoring for the modifiedprotein into the cell membrane for optimal interaction with its respective effector proteins. FPP, farnesylpyrophosphate; FTase, farnesyl transferase; CMT, carboxyl methyl transferase; PMT, palmitoyl transferase.

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wise, addition and deletion of palmitoyl groups arefacilitated by palmitoyl transferase and esterase, re-spectively (58). Studies from our laboratory (2, 3537,39, 4153, 55, 65, 69) and those of others (34, 54, 56,83) have demonstrated the requisite nature and rolesof posttranslational modifications of these proteins inphysiological insulin secretion. They are discussed inthe following sections.

Islet G Protein Prenylation and Insulin Secretion

Using generic as well as more specific inhibitors (seeTable 1), numerous earlier studies have demonstratedcritical regulatory roles for protein prenylation inphysiological insulin secretion and identified some ofthese proteins as Cdc42, H-Ras, -subunits of trimericG proteins, and the nuclear lamin-B (2, and see Ref. 48for a review). Needless to say, this list is only partial.Initial studies that examined possible roles of proteinprenylation in islet function utilized statins (56, 69,106), as they inhibit the synthesis of MVA, a precursorfor the biosynthesis of isoprenoid derivatives (e.g., far-

nesyl or geranylgeranyl pyrophosphates), which areincorporated into respective proteins to complete theisoprenylation step (Fig. 1). Preincubation of isolatednormal rat islets or clonal -cells with lovastatin hasbeen shown to result in selective accumulation of non-prenylated proteins in the soluble fraction, with a con-comitant decrease in their abundance in the membranefraction. Under these conditions, lovastatin signifi-cantly inhibited glucose-stimulated insulin secretionfrom normal rat islets (69) (Fig. 2) and from bombesin-

and vasopressin-mediated insulin secretion HIT-T15cells (56). Even though the identity of all of the Gproteins critical for this process has not been deter-mined, indirect evidence suggests that Cdc42 mightrepresent one such protein. For example, in trans-formed -cells, lovastatin reduces prenylation of Cdc42and thereby impedes its complexing with a GDP-disso-ciation inhibitor (83). This, in turn, leads to its redis-

tribution from membranes to cytosol, effects not seenwith some other monomeric G proteins [e.g., Rho or ADP ribosylation factor (ARF)]. Together, data fromthese studies indicate that inhibition of protein preny-lation in -cells results in selective accumulation ofunprenylated G proteins in the soluble compartment,possibly interfering with the interaction of these pro-teins with their respective effector proteins, which maybe required for nutrient-induced insulin secretion.Data from studies using more generic inhibitors ofprotein isoprenylation (e.g., limonene, perillic acid; seeTable 1) were not very conclusive because of theirnonspecific and cytotoxic effects on islet function (39,56, 69).

Recently, we synthesized a novel class of prodruginhibitors, such as 3-allyl and vinyl-farnesols and 3-al-lyl and 3-vinyl geranylgeraniols, which inhibited (witha greater specificity) the protein farnesyl and gera-nylgeranyl transferases, respectively. These twoclasses of inhibitors significantly reduced glucose- andcalcium-stimulated insulin secretion from -TC3 cells(2). The degree of inhibition was much greater thanwhat was demonstrable in the presence of lovastatin inisolated rat islets, suggesting that they are much moresite specific than the classical hydroxymethyl glutaryl-CoA reductase blockers (Table 1). Akin to lovastatin,allyl and vinyl farnesols and geranylgeraniols signifi-

cantly influenced the subcellular distribution of smallG proteins, as evidenced by a considerable degree ofaccumulation of the unprenylated proteins in the cyto-solic fraction, with a concomitant decrease in theirabundance in the membrane fraction (2). Together,these cited studies indicate that protein prenylationplays a significant regulatory role in physiological in-sulin secretion. It is also apparent that a substantialamount of work is still needed, especially in the area ofidentification of these prenylated proteins, as well asthe prenylating enzymes (e.g., isoprenyl transferases).Recent evidence from our laboratory suggests immu-nological localization of farnesyl and geranylgeranyltransferases in insulin-secreting cells (40). As we have

pointed out, even though protein prenylation is notacutely regulable, it seems to dictate the subsequentmodification steps (e.g., carboxyl methylation) that areacutely regulated and to determine the functional sta-tus of a given G protein.

Islet G Protein Methylation and Insulin Secretion

Unlike protein prenylation, the carboxyl methyl-ation of prenylated cysteine is acutely regulable, andboth the methylating and demethylating enzymes havebeen characterized in mammalian cells, including the

Fig. 2. Inhibitors of posttranslational modifications of small G pro-teins markedly reduce glucose-stimulated insulin secretion fromnormal rat islets. Effects of inhibitors of prenylation [e.g., lovastatin(LOVA); 15 M], carboxyl methylation [e.g., acetyl farnesyl cysteine(AFC); 100 M], or palmitoylation [e.g., cerulenin (CER); 134 M] onglucose (16.7 mM)-stimulated (control) insulin secretion from normalrat islets are shown as indicated. Representative data from studiesdescribed in our earlier publication (69) were plotted in this figure.Data indicate a marked attenuation by inhibitors of all 3 classes ofrequisite modifications of small G proteins of glucose-stimulatedinsulin secretion from normal rat islets. *P 0.001. (See Table 1 fora summary of observations from various laboratories on potentialregulation by these modification steps of insulin secretion elicited by

various insulin secretagogues in different insulin-secreting celltypes.)

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pancreatic -cell (39, 41, 55). The carboxyl methyltransferase catalyzes the incorporation of a methylgroup onto the carboxylate anion of the prenylatedcysteine via an ester linkage. It utilizes intracellularS-adenosyl methionine (SAM) as the methyl donor.Several studies, including our own, have identifiedcarboxyl-methylated proteins in the pancreatic -cell.These include Cdc42, Rap1, Rac 1, H-Ras, the -sub-

units of trimeric G proteins, and the nuclear lamin-B(35, 41, 49, 54, 95).

A previous study characterized the prenyl cysteinemethyl transferase activity in insulin-secreting cellsand normal rat islets (55). This activity was monitoredby quantitating the degree of methylation of an artifi-cial substrate [e.g., acetyl farnesyl cysteine (AFC)] with[3H]SAM as methyl donor. Subcellular fractionationstudies revealed that this enzyme is localized in theplasma membrane and the endoplasmic reticular frac-tions. Even though several lines of experimental evi-dence indicate that the carboxyl methylation of specificG proteins (e.g., Cdc42 and Rap1) is stimulated byexogenous GTP (49, 54), we observed that exogenous

GTP had no demonstrable effect on this enzyme, sug-gesting that this enzyme may be constitutively activewithin the -cell, and that the methylation of targetproteins in vivo is regulated by the access of theseproteins to the methyl transferase, as well as theiractive GTP-bound conformation (55). It may be ger-mane to point out that, in addition to the carboxylmethylation at COOH-terminal cysteine, we reportedmethylation of COOH-terminal leucine, especially ofthe catalytic subunit of protein phosphatase 2A(PP2Ac) (51). Inhibitors of protein phosphatases, suchas okadaic acid, inhibited the carboxyl methylation ofPP2Ac. Data derived from the inhibitor experiments

provide useful insights into the applicability of inhibi-tors of protein carboxyl methylation for study of puta-tive roles of different proteins in cellular regulation.For example, AFC inhibits the methylation at aCOOH-terminal cysteine, whereas okadaic acid specif-ically inhibits the carboxyl methylation of COOH-ter-minal leucine (41, 49, 51, 54).

Several earlier investigations have examined therelevance of prenyl cysteine carboxyl methylation inglucose-induced insulin secretion (49, 69) (see Fig. 2).For example, by use of rat islets and clonal -cells,glucose has been shown to stimulate the carboxylmethylation of Cdc42 and Rap1 in a transient manner.Stimulation of carboxyl methylation of these proteins

was demonstrable within 1530 s after exposure ofcells to glucose (49). It was also shown that such anincrease in the carboxyl methylation of these proteinswas specifically blocked by AFC, because a structurallysimilar inactive analog of AFC, namely, acetyl gera-nylgeranyl cysteine (AGGC), was without any effect.Studies from Fleischers group (Leiser et al., Ref. 54)have also utilized these specific probes to determinethe relative contribution of Rap 1, another monomericG protein, in glucose- and calcium-mediated insulinsecretion. Follow-up studies from our laboratory have

utilized similar experimental approaches and probes todecipher the roles of the carboxyl methylation of the-subunits of trimeric G proteins in glucose-mediatedinsulin secretion (41).

Finally, by use of specific inhibitors of GTP biosyn-thesis [e.g., mycophenolic acid (MPA)], it was possibleto establish a critical requirement for endogenous GTPin glucose-stimulated carboxyl methylation of specific

G proteins and concomitant stimulation of insulin se-cretion from isolated rat islets (39, 41, 49). Depletion ofendogenous GTP markedly reduced the ability of glu-cose to stimulate the carboxyl methylation of specificislet proteins (e.g., Cdc42, G-subunits of trimeric Gproteins) as well as insulin secretion, suggesting thatendogenous GTP is essential for these signaling stepsleading to insulin secretion (39, 41, 49). Such a formu-lation was further supported by additional observa-tions indicating that provision of guanosine exog-enously to GTP-depleted cells completely reversed theability of glucose to activate the carboxyl methylationof these two proteins, as well as insulin secretion. Thereversal effects appear to be specific for guanosine,since exogenous adenosine failed to reverse the inhib-itory effects demonstrable after GTP depletion (39, 41,49). These data indicate a clear dependence of endog-enous GTP in physiological insulin secretion, presum-ably mediated by the activation of trimeric as well asmonomeric G proteins. The reader is referred to Table1 for a summary of findings from various laboratorieson the effects of inhibitors of protein carboxyl methyl-ation on insulin secretion from isolated -cells.

Islet G Protein Palmitoylation and Insulin Secretion

As indicated in Fig. 1, fatty acids (typically, palmi-tate) are incorporated posttranslationally into specificG proteins via a thioester linkage at cysteine residuesupstream of the prenylated and methylated cysteine(48, 92, 103). This modification is thought to furtherfacilitate the interaction of G proteins with their mem-brane-bound effectors. Several previous studies indi-cated that the -subunits of trimeric G proteins may beacylated; this is regulated acutely in response to recep-tor activation, thereby controlling the subcellular dis-tribution of these -subunits (i.e., membrane vs. cyto-solic). Receptor activation has also been shown to reg-ulate protein deacylation (103). Cerulenin, a selectiveblocker of protein acylation, has been shown to reducenutrient-induced insulin secretion from isolated rat

islets (69) (Fig. 2); these data were further confirmedalso in normal rat islets by Yajima et al. (109). Inter-estingly, cerulenin failed to inhibit insulin secretionfacilitated by nonnutrient secretagogues, such as amembrane-depolarizing concentration of potassium,activators of protein kinase A, or mastoparan. To-gether, these data support a critical regulatory role forprotein acylation steps in -cell function. It may bementioned that the inhibitory effects of cerulenin (spe-cifically, at higher concentrations and over longer pe-riods of incubation) on protein acylation are rather

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nonspecific, because this probe can inhibit fatty acid,sterol, and protein synthesis. 2-Bromopalmitate hasalso been used to study the roles of protein acylation in

cellular function (102). More specific cerulenin analogshave been reported recently (13) and await furtherinvestigations. Interestingly, experimental and struc-tural data indicate that certain proteins, which un-dergo prenylation as well as carboxyl methylation (e.g.,Cdc42 or -subunits of trimeric G proteins), are notsubject to fatty acylation (see Ref. 48 for a review).Therefore, it is likely that acylation of -subunits oftrimeric G proteins and/or other low-molecular-weightG proteins (e.g., Ras) may also be necessary for insulinsecretion. Alternatively, other proteins involved in theexocytotic process, such as SNAP-25 (18, 97), may becritically acylated. Additional studies are needed todemonstrate conclusively a putative role(s) for fatty

acylation, as well as the identity of candidate G pro-teins in physiological insulin secretion.

Use of Clostridial Toxins To Examine the Roleof G Proteins in Insulin Secretion

Several lines of evidence suggest that clostridial tox-ins serve as extremely useful tools to study putativeregulatory roles of the Rho subfamily of G proteins incellular function (39, 42, 49, 86). These toxins specifi-cally monoglucosylate and inactivate G proteins withreliable specificity (Table 2). For example, Clostridiumdifficile toxins A or B monoglucosylate (at threonineresidues) Rho, Rac, and Cdc42 (but not Ras, Rab, orARF) proteins; this modification impairs the functionof these small G proteins. Clostridium sordellii lethaltoxin monoglucosylates Rac, Rap, and Ras specifically,

Table 1. Known effects of inhibitors of posttranslational modifications of G proteins on insulin secretion

Type of Modification Observation Ref.

PrenylationLovastatin Significantly inhibited (46 to 57%) glucose-stimulated insulin secretion from normal

rat islets. No significant to minimal effects on phorbol ester-, high potassium- or-oxo-4-methyl-pentanoic acid-induced insulin secretion

69

Inhibited potentiating effects by bombesin and vasopressin of nutrient-inducedinsulin secretion from HIT-T15 cells. However, potentiating effects by phorbol ester

or forskolin were unaffected

55

Simvastatin Significantly inhibited glucose-stimulated insulin secretion from single -cells andnormal rat islets via inhibition of L-type calcium channels. Simvastatin acid, a lesslipophilic inhibitor, was less potent

106

Pravastatin No effect on L-type calcium channels and glucose-induced insulin secretion, probablydue to its hydrophilicity

106

Allyl and vinyl farnesols andgeranylgeraniols

Significantly inhibited glucose- and calcium-induced secretion from TC3 cells 2

GGTI-2147 Significantly inhibited glucose- and calcium-induced secretion from TC3 cells 2Manumycin Significantly inhibited glucose- and calcium-induced secretion from TC3 cells 2

Methylation Acetyl farnesyl cysteine Acute exposure to rat islets attenuated glucose-stimulated (40 to 60%) and -oxo-4-

methyl-pentanoate-induced (68 to 85%) insulin secretion. Acetyl geranyl cysteine(AGC), an inactive analog of AFC, had no effect on glucose-stimulated insulinrelease. AFC had no effect on mastoparan- or high potassium-stimulated insulinsecretion

69

Acetyl geranylgeranyl cysteine Stimulated basal, calcium- or GTP-induced insulin secretion from streptolysin-O

permeabilized HIT-T15 cells. AFC was less potent and AGC was inactive

84

Homocysteine plus deazaadenosine More global inhibitors of methylation. Potently inhibited glucose-stimulated (35%)or amino acid-induced (62%) insulin secretion

69

AcylationCerulenin Significantly reduced fractional rates of insulin secretion stimulated by glucose (63

to 88%), amino acid-induced (73 to 100%), but not mastoparan-induced insulinsecretion from isolated rat islets

69

Significantly inhibited both phases of glucose-induced, but not potassium-induced,insulin secretion from isolated rat islets

91

Table 2. Specificity of bacterial toxins used for addressing the roles of small G proteinsin stimulus-secretion coupling of the islet -cell

Toxin Used Type of Modification Target G Protein(s) Effect on Function

Clostridium difficile Glucosylation Rho, Rac, and Cdc42 InactivationClostridium sordellii Glucosylation Rac, Rap, and Ras InactivationClostridium novyi Glucosaminylation Rho, Rac, and Cdc42 InactivationClostridium C3-exoenzyme Ribosylation Rho InactivationCytotoxic necrotizing factor Deamidation Rho Activation

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but not Cdc42, Rho, or Rab. In recent years, clostridialtoxins have been used to seek further support for theabove formulation that Rho proteins (e.g., Cdc42 andRac) are involved in -cell signal transduction. Expo-sure of normal rat islets or clonal -cells to C. difficiletoxin A or B significantly reduced glucose-induced in-sulin secretion. These data indicated that Rac, Cdc42,and Rho G proteins are involved in this phenomenon

(42). Interestingly, C. sordellii toxin also reduced glu-cose-induced insulin secretion from these cells undersimilar experimental conditions, suggesting that Ras,Rap, and Rac are also involved in this phenomenon. C3exoenzyme, which ADP ribosylates and inactivatesRho, failed to inhibit glucose-induced insulin secretionfrom these cells, suggesting that Rho may not be involved in this process (42). Together, these findingshave led to the conclusion that Cdc42, Rap, Rac (allgeranylgeranylated proteins), and Ras (a farnesylatedprotein) might be involved in physiological insulin se-

cretion. These findings are compatible with our obser-vations using allyl farnesols and geranylgeraniols (2).

Use of Mastoparan to Examine the Role of G Proteinsin Insulin Secretion

Mastoparan (Mas), a tetradecapeptide from wasp venom, has been shown to activate a wide variety of

heterotrimeric as well as small G proteins, presumablyby facilitating GTP/GDP exchange (21, 22). Severalearlier studies have demonstrated that Mas stimulatesinsulin secretion from normal rat islets, human islets,and clonal -cells (see Table 3 for a summary of thesestudies). However, the precise loci for Mas regulationof insulin secretion remain less understood. Recentevidence from our laboratory suggested that Mas-in-duced insulin secretion from isolated -cells involvesactivation of Rac (3). Further experiments indicatedthat Mas activates Rac via GTP/GDP exchange but not

Table 3. Summary of data from earlier studies that used mastoparan to study stimulus-secretioncoupling in the islet -cell

Cell Type Studied Observation Ref.

Rat pancreatic islet Ptx- or bromophenacyl bromide, a PLA2 inhibitor, abolished Mas-stimulatedinsulin secretion

110

Rat pancreatic islet Ptx or neomycin, an inhibitor of PLC, blocked Mas-stimulated insulin secretion.Nifedipine, somatostatin, inhibitors of PKA or PKC, had no demonstrableeffects

31

RINm5F cells Ptx or Ctx treatment had no demonstrable effects on Mas-induced insulinsecretion

23

Intact or permeabilized rat islets Mas caused temperature-dependent insulin secretion. Extracellular calciumwas not necessary. PKC or cAMP antagonists had no effects. Inhibited byGDPS

26

Rat pancreatic islets Mas-induced insulin secretion was unaffected by inhibitors of posttranslationalmodifications of G proteins, including lovastatin and acetyl farnesyl cysteine

69

RINm5F cells Ptx pretreatment enhanced insulin secretion induced by Mas 32Rat islets and human islets Mas stimulated a high-af finity GTPase activity in the secretory granule fraction 46Rat islets, human islets, HIT-T15 cells

and rat insulinoma cellsMas stimulated nucleoside diphosphate kinase (NDPK) activity. Interestingly,

Mas-17 an inactive analog of Mas, also stimulated NDPK activity43, 50

TC3 cells Mas analogs, but not Mas-17, stimulated insulin secretion in a Ptx-sensitivemanner. Mas also stimulated a GTPase activity associated with insulinsecretory granules

34

RINm5F cells In contrast to glyceraldehyde-, A-23187-, or carbachol-induced insulin secretion,Mas-stimulated insulin release was unaffected by pancreastatin

20

Normal rat islets, human islets andclonal -cells

Mas, but not Mas-17, stimulated P-His phosphorylation in the membrane andsecretory granule fractions

52

Normal rat islets and islets from theGoto-Kakizaki rat

Galparan, a peptide consisting of galanin (113 residues) and Mas, stimulatedinsulin secretion from control and diabetic rat islets. Stimulatory effects ofgalparan were insensitive to Ptx pretreatment

76

Rat and human pancreatic islets Mas-stimulated insulin secretion in the absence of extracellular calcium. Underthese conditions, it also augmented glucose-simulated secretion. Both effectsof Mas were Ptx insensitive

90

Normal rat islets and islets from theGoto-Kakizaki rat Unlike abnormalities in glucose-, calcium-, or mitochondrial fuel-inducedinsulin secretion, Mas-stimulated secretion was completely normal in thediabetic GK rat islets

67

Rat pancreatic islets Mas-stimulated insulin secretion was not affected by cerulenin, an inhibitor ofprotein acylation

109

MIN6 cells Mas-stimulated release of insulin and GABA. Overexpression of syntaxin 1Aand SNAP-25 markedly reduced Mas-stimulated insulin release from thesecells

74

Insulin-secreting HC-9 cells Overexpression of Cdc42 markedly increased Mas-stimulated insulin secretionin these cells in a Ptx-independent manner

11

Normal rat islets, human islets, andclonal -cells

Mas, but not Mas-17, its inactive analog, stimulated a novel histone-4phosphorylating histidine kinase activity

37

INS-1 cells Expression of dominant negative mutant of Rac1 (N17 Rac1) markedlyattenuated mas-induced insulin secretion

3

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via modulation of its isoprenylation. Transfection ofdominant negative Rac (N17 Rac) markedly attenu-ated Mas-induced (3) or glucose- and forskolin-induced(57) insulin secretion from clonal -cell preparations,suggesting that Rac plays an important role in insulinsecretion elicited by different secretagogues. Recentinvestigations by Daniel et al. (11) have also identifiedCdc42 as one of the proteins involved in Mas-stimu-

lated insulin secretion. Mas and Mas-17 (its inactiveanalog) are also proven to be as valuable probes inrecent studies (Fig. 3), which addressed the insulin-secretory abnormalities in islets derived from theGoto-Kakizaki (GK) rat, a model for non-insulin-dependent diabetes mellitus (NIDDM) (67). We re-ported that, whereas glucose- and potassium-in-duced insulin secretion was reduced significantly inislets from the GK rat, the Mas-induced insulinsecretion remained unaltered in these islets. In GKislets, we also observed significant defects in thefunctional activation of nucleoside diphosphate ki-nase (NDPK), and on the basis of these data weproposed that the abnormalities in insulin secretion

in the GK rat may lie at the level of an NDPK-mediated Mas-sensitive G protein (see the followingsections for a summary of these and other relatedstudies).

When the evidence just described is considered asa whole, it is evident that the Rho subfamily of Gproteins play critical regulatory roles in physiologi-cal insulin secretion. However, it must be kept inmind that most, if not all, studies that were citedabove (and in Tables 1 and 2) utilized chemicalinhibitors of the requisite posttranslational modifi-cations (e.g., statins) or bacterial toxins (e.g., clos-tridial toxins). Such approaches are often ques-

tioned for the nonspecific nature of the chemicalinhibitors and toxins used to arrive at the respectiveconclusions. Although the degree of specificity ofthese probes was well studied and described, defini-

tive support to the extant studies and further proofof potential regulatory roles for these G proteins inphysiological insulin secretion must also be verifiedby gene depletion approaches. At the outset, at leastthree members of the Rho subfamily of G proteins,namely Cdc42, Rap1, and Rac1, must be given seri-ous consideration for gene depletion studies to assesstheir contributory roles in physiological insulin se-

cretion from the isolated -cell. This suggestion isbased on the following experimental evidence. First,we (41, 49) and others (54) have demonstrated thatCdc42, Rac1, and Rap1 undergo glucose- and calci-um-mediated carboxyl methylation and subsequentactivation in normal rat islets and clonal -cells.Second, clostridial toxins, with defined specificity forinactivation of these proteins, markedly reduced glu-cose- and calcium-mediated insulin secretion (42).Third, novel geranylgeranyl transferase inhibitors,with the highest degree of specificity to inhibit theseproteins, markedly reduced glucose-and calcium-in-duced insulin secretion from -cells (2). Taken to-

gether, these data assign major regulatory roles forCdc42, Rac1, and Rap1 in physiological insulin se-cretion. More recent molecular biological data alongthese lines tend to further support a role for these Gproteins in insulin secretion. Daniel et al. (11) re-ported a marked stimulation in Mas-induced insulinsecretion in -cells after expression of Cdc42. Usinga dominant negative mutant for Rac1 (N17 Rac1), werecently reported significant inhibition in Mas-in-duced (3) and glucose- and forskolin-induced (57)insulin secretion from isolated -cells. Althoughthese data are encouraging, additional studies areneeded to further verify the putative regulatory rolesof these signaling proteins in insulin secretion, spe-

cifically via gene depletion approaches.Together, on the basis of information reviewed

above, it is clear that activation of certain G proteins,specifically those belonging to the Rho subfamily, isimportant for insulin secretion elicited by glucoseand other secretagogues in the -cell. It is also be-coming increasingly evident that abnormalities inthe activation of specific G proteins could contributeto alterations in the insulin secretion demonstrablein models of impaired insulin secretion (see the fol-lowing sections). The fundamental question of howglucose (and other insulin secretagogues) activatethe islet endogenous G proteins still remains unan-

swered at this time. Along these lines, we (41, 42, 49)and others (54) have provided experimental evidenceto indicate that glucose augments posttranslationalmodifications (e.g., carboxyl methylation) of specificG proteins (e.g., Cdc42 and Rap1) in a GTP-sensitivemanner. In addition to these possibilities, and on thebasis of more recent data obtained in our laboratory,I propose that activation of candidate G proteins byglucose may be mediated via the transphosphoryla-tion of GDP bound to G proteins (inactive conforma-tion) to their GTP-bound active conformation throughthe intermediacy of novel protein histidine kinases

Fig. 3. Structure-specific stimulation by mastoparan (Mas) of insulinsecretion from isolated rat islets. Insulin release was measured fromfresh, isolated normal rat islets in static incubation conditions at 3.3mM glucose. Thirty micromoles each of Mas or Mas-17 (an inactiveanalog of Mas) were present during the 45-min incubation period, asindicated. Representative data from studies described in our earlierpublication (43) were plotted. Data represent means SE from 35determinations in each case. *P 0.001 vs. control.

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that we have recently identified in the islet -cell (seeTable 4).

NOVEL REGULATORY MECHANISMS FOR THE

ACTIVATION OF G PROTEINS IN THE ISLET -CELL:

EVIDENCE FOR THE INVOLVEMENT OF PROTEIN

HISTIDINE PHOSPHORYLATION

In most cells, the transduction of extracellular sig-nals involves ligand binding to a receptor, often fol-lowed by the activation of one (or more) G proteins andtheir effector systems (6, 17). The pancreatic -cell isunusual in that glucose, the major physiological ago-nist, lacks an extracellular receptor. Instead, eventsconsequent to glucose metabolism promote insulin se-cretion via the generation and/or altered distribution ofdiffusible second messengers, such as ions, cyclic nu-cleotides, and biologically active lipids (44, 48, 59, 60,73, 81). Changes in calcium concentration not onlyinitiate insulin secretion but also regulate various en-zymes, such as protein kinases, phosphodiesterases,adenylyl cyclases, and phospholipases, thereby facili-tating insulin secretion. In addition to calcium-depen-dent protein kinase(s), several other kinases, includingcalmodulin-, cyclic nucleotide-, phospholipid-depen-dent protein kinases, tyrosine kinases, and mitogen-activated protein kinases have been described in-cells (see Ref. 27 for a review). The majority of thesekinases mediate phosphorylation of endogenous -cellproteins using ATP as the phosphoryl donor. In addi-tion, we (52) reported evidence for the localization of anovel protein kinase in -cells that selectively usesGTP as a phosphoryl donor and uniquely phosphory-lates specific proteins (e.g., -subunit of trimeric G

proteins) at histidine residues. We (52) further demon-strated that this phosphate, in turn, is transferred tofree GDP (or GDP liganded to G proteins) to yield freeGTP (or GTP bound to G proteins).

Protein Histidine Kinases

To date, the most phosphorylated amino acids iden-tified include serine (P-Ser), threonine (P-Thr), andtyrosine (P-Tyr). Phosphoamino acids exhibit differen-tial sensitivities to acidic and alkaline pH conditions(62). P-Ser and P-Thr, which form O-p (alcoholic O-monoester) linkages, are stable at acidic pH and are

fairly unstable under alkaline conditions. P-Tyr, whichforms O-p (phenolic O-monoester), is stable underacidic and alkaline conditions. Therefore, because oftheir stability under acidic conditions, P-Ser, P-Thr,and P-Tyr are readily identified after acid hydrolysis ofphosphorylated proteins. However, acid-labile phos-phoramidate linkage has been reported (62, 63) inhistidine (P-His), arginine (P-Arg), and lysine (P-Lys).

It is not surprising that very little information is avail-able on the number of proteins with P-His, since itsphosphate is rapidly lost during identification of phos-phoamino acids under standard acid hydrolysis condi-tions or under conditions used for SDS-PAGE (52, 62,63, 104). It is estimated that P-His may account for 6%of total protein phosphorylation in eukaryotes. In thiscontext, it has also been shown that P-His undergoesrapid dephosphorylation in crude cellular extracts (28,30), including pancreatic islet cell lysates, as we re-ported in Ref. 52.

Several recent studies have investigated protein his-tidine phosphorylation in multiple cell types. For ex-

ample, Huang et al. (25) purified a monomeric histidinekinase from Saccharomyces cerevisiae with an appar-ent molecular mass of 32 kDa. This kinase exhibitedspecificity toward ATP (also GTP, but with minimalaffinity) to phosphorylate histone-4. This enzyme re-quired divalent cations for maximal activity; spermineor spermidine was ineffective. Motojima and Goto (70)reported histidine phosphorylation of a 36-kDa proteinby a histidine kinase in liver extracts. They also re-ported localization of an okadaic acid-resistant phos-phatase activity (with an apparent molecular mass of45 kDa). Using an HPLC method, they demonstratedcopurification of the kinase and p36 substrate at a 70-to 75-kDa size. These data indicate that the liver his-

tidine kinase may be different from the yeast enzymeoriginally described by Huang et al. Along similarlines, Urushidani and Nagao (96) also reported auto-phosphorylation, at a histidine residue, of a 40-kDaprotein localized in the membrane fraction derivedfrom rabbit gastric mucosa. Sequence analyses dataindicated that this protein might represent the -sub-unit of an extramitochondrial isoform of succinyl-CoAsynthetase (SCS) or its homolog. Autophosphorylationof this protein was stimulated by GDP, Ras (a small Gprotein), and myelin basic protein and was rapidlydephosphorylated in the presence of ATP, succinate,and CoA. Hegde and Das (19) showed that Ras stimu-

lated the phosphorylation of a 36-kDa protein at ahistidine residue in liver membranes. More recently,Besant and Attwood (5) purified and characterized ahistone 4-phosphorylating histidine kinase activityfrom porcine thymus. This enzyme appears to havecertain similarities with the yeast enzyme, includingthe molecular mass, which was estimated to be 3441 kDa. Together, these studies identified localizationof a histidine-phosphorylating enzyme(s) that appearsto be regulated under various experimental conditions(e.g., in the presence of Ras). The reader is referred toseveral recent reviews (1, 30, 71, 79, 89, 93) that

Table 4. Known histidine kinases, their potentialphosphoprotein substrates, and their subcellularlocalization in insulin-secreting cells

Proteins Localization Phosphorylating Kinase

NDPK (nm 23-H1and nm-23-H2)

Cytosol and membrane Autophosphorylation

NDPK (nm 23-H4) Mitochondria Autophosphorylation-subunit of

trimeric Gproteins

Membrane andsecretory granules

Histidine kinase

Succinyl-CoAsynthetase (ATPand GTPspecific)

Mitochondria NDPK

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describe potential regulatory roles of various histidinekinases in cellular regulation and function.

Using SDS-PAGE and the nitran filter paper assay,we (37) recently characterized a protein histidine ki-nase in the lysates of normal rat islets, human islets,and clonal -cell (HIT-T15 and INS-1) cell prepara-tions. The -cell histidine kinase is sensitive to ATP aswell as GTP, with an apparent molecular mass of

6070 kDa. Noticeable similarities appear to exist be-tween the -cell and the yeast histidine kinases. Forexample, both use ATP as well as GTP as phosphoryldonors, and both enzymes exhibit similar metal ionrequirements and were resistant to polyamines. Theprincipal difference appears to be the size of the en-zyme. The -cell enzyme is 6070 kDa in size incontrast to the yeast enzyme, which has been shown tobe 32 kDa. On the basis of our additional observa-tions in the -cell, we suggest that phosphohistidinephosphorylation may be important in insulin exocyto-sis from the -cell. In support of this formulation, wedemonstrated (37) that the -cell histidine kinase isactivated in a structure-specific manner by Mas. Mas

or Mas-7, but not Mas-17 (an inactive analog), is apotent activator of insulin secretion (Table 2). We ob-served similar specificities for the activation by Masanalogs of histidine kinase activity, as well as the-subunit phosphorylation and insulin secretion, in ratislet homogenates (37). Although several previousstudies, including our own (see Table 3), have demon-strated insulinotropic effects of Mas, our data suggestfor the first time that Mas-mediated signaling eventscould include activation of protein histidine phosphor-ylation in the pancreatic -cell. Furthermore, these

data establish a biochemical link between activation ofhistidine kinase and activation of phosphorylation ofthe -subunit through the use of Mas, a global Gprotein activator. Additional studies are needed to un-derstand precisely the regulation of this enzyme bynutrient insulin secretagogues and G protein-coupledreceptor agonists to conclusively establish a link be-tween activation of G proteins (via activation of this or

other related histidine kinases) and insulin secretionfrom isolated -cells. On the basis of our data onhistidine kinase-mediated phosphorylation of the-subunit of trimeric G proteins, we propose a modelfor the activation of trimeric G proteins in the -cellinvolving protein histidine phosphorylation (Fig. 4).We propose that physiological insulin secretagogues(e.g., glucose) elicit effects on functional activation ofspecific G proteins via receptor-independent mecha-nisms. Our model predicts that glucose and other nu-trient secretagogues stimulate histidine phosphoryla-tion of specific transmitter proteins (e.g., the -sub-unit of trimeric G proteins) and that this phosphate, inturn, is transferred to a receiver protein, such as the-subunit (in its GDP-bound inactive conformation) toyield its GTP-bound active conformation. In support ofour hypothesis that cellular metabolism leads to rapidprotein histidine phosphorylation, Crovello et al. (8)provided the first direct evidence for the induction ofrapid and reversible histidine phosphorylation inmammalian cells upon activation. Using human plate-lets, they demonstrated transient phosphorylation ofP-selectin at a histidine residue by thrombin or colla-gen. Although the activation mechanism proposed inFig. 4 pertains to trimeric G proteins, it is also likely

Fig. 4. Proposed mechanism for receptor-independent activation of trimeric G proteins in the pancreatic -cell byglucose. Trimeric G proteins remain inactive when their -subunit is bound to GDP. We propose that a histidinekinase phosphorylates the -subunit of trimeric G proteins at a histidine residue via a phosphoramidate linkage.This phosphate in turn is relayed to the GDP-bound -subunit and transphorylates the GDP to GTP. Then, the-subunit bound to GTP dissociates from the -complex for regulation of its effector proteins. Ample experimentalevidence identified multiple effector proteins for the -subunits as well as the -complex in several cellularsystems. After hydrolysis of GTP by GTPase activity intrinsic to the -subunit, -GDP reassociates with the-complex to complete one activation cycle. Not shown here is the possibility of nucleoside diphosphate kinase(NDPK) subserving the role of histidine kinase in mediating the phosphorylation of the -subunits (see text foradditional details).

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that similar activation mechanisms are operable in thecontext of small G proteins. This may be mediated viathe NDPK-catalyzed reaction. In the following sec-tions, we propose a model (Fig. 5) that predicts nutri-ent-mediated regulation of NDPK, which in turn gen-erates GTP in the vicinity of candidate small G pro-teins necessary for their activation. Alternatively,NDPK could subserve the function of transphosphory-

lating the GDP bound to G proteins (i.e., their inactiveconformation) to their GTP-bound, active conforma-tion.

NDPK

The enzyme NDPK catalyzes the transfer of terminalphosphates from nucleoside triphosphates (e.g., ATP)to nucleoside diphosphates (e.g., GDP) to yield theirrespective nucleoside triphosphates (e.g., GTP). Thetransfer of terminal phosphates occurs by a two-step,ping-pong reaction involving the formation of a tran-sient high-energy phosphoprotein intermediate form ofNDPK, due to phosphorylation at a histidine residue,

followed by transfer of that phosphate to a suitableacceptor (29). In addition to the generation of nucleo-side triphosphates, NDPK has been implicated in thedirect activation of certain G proteins as well as phos-phorylation and/or regulation of several key enzymesof intermediary metabolism (e.g., ATP citrate lyase,aldolase, pyruvate kinase, glucose-6-phosphatase, andSCS) (15, 53, 89, 96, 99, 100).

Although multiple roles have been described forNDPK [the reader is referred to recent reviews on

NDPK describing potential regulatory roles of thisenzyme in regulation of cellular function (29, 89)], oneof the unique roles of NDPK (in the context of thiscurrent review and -cell metabolism) is its ability tocontribute toward the synthesis of GTP and the subse-quent activation of specific G proteins. The latter isthought to occur via chaneling of GTP to the vicinityof candidate G proteins for their functional activation.

It has also been shown that NDPK mediates trans-phosphorylation of GDP bound to G proteins (inactiveconformation) to their GTP-bound (active conforma-tion) of G proteins (43). Original studies from ourlaboratory (43) have characterized NDPK activity innormal rat and human islets as well as clonal -cellpreparations. More recent studies (53) have identifiedat least three isoforms of NDPK in the pancreatic-cell. They include nm23-H1, a predominantly mem-brane-associated form of NDPK, and nm23-H2, with amembranous as well as soluble localization. In addi-tion, a mitochondrial isoform of NDPK (nm23-H4) hasbeen identified in the islet -cell (53). Potential roles ofthese isoforms and significance of their subcellular

distribution have also been described in Ref. 53. On thebasis of our current understanding of the biochemicalproperties and physiological regulation of this enzymein the islet -cell, we propose a model for potentialcontributory roles of NDPK in glucose-stimulated in-sulin secretion, specifically at the level of activation ofG proteins (Fig. 5). We propose that glucose-inducedincreases in the GTP/GDP ratio (as demonstrated ear-lier in Refs. 12 and 66) may in part be due to theactivation of NDPK, which generates GTP via trans-phosphorylation of GDP from ATP. This increase inGTP concentrations favors either increase in GTP/GDPexchange on a relevant G protein[s] or chaneling of

GTP to candidate G protein(s), culminating in theiractivation. In addition, it is likely that glucose alsoactivates the histidine kinase (as described in the pre-vious section), resulting in stimulation of the phosphor-ylation of key regulatory proteins, including the -sub-units of trimeric G proteins at a histidine residue. Sucha phosphate, in turn, is transferred to the GDP boundto the -subunits of trimeric G proteins via the phos-pho-relay mechanism (52, 71, 79, 105) that is given inFig. 4. We also propose that glucose-mediated activa-tion of NDPK might result in histidine phosphorylationof other proteins, such as SCS, aldolase, and ATP-citrate lyase, which is required for their functionalactivation, and subsequent insulin secretion. For ex-

ample, SCS catalyzes the substrate level phosphoryla-tion of ADP or GDP. In the context of SCS regulation inthe islet -cell, we have recently shown that the -sub-unit of SCS undergoes phosphorylation at a histidineresidue, which may be catalyzed by NDPK-mediatedphosphotransfer mechanisms (36, 53). In support ofthis, we have demonstrated colocalization as a complexof mitochondrial NDPK and SCS in the -cell mito-chondria. Using the mitochondrial extracts from clonal-cells (INS-1 and HIT-T15), we have been able toquantitate the formation of succinyl-CoA from succi-nate, CoA, and ATP or GTP. Furthermore, using im-

Fig. 5. Proposed mechanisms for glucose-stimulated activation of Gproteins involving members of the histidine kinase family. We pro-pose that, in addition to increasing GTP biosynthesis, glucose acti-

vates NDPK to facilitate transphosphorylation of GDP to GTP. Suchan increase in GTP levels, specifically in the vicinity of candidate Gproteins, results in activation of those G proteins, leading to stimu-lation of insulin secretion (left). On the basis of recent data (reviewedin the text), it is also likely that NDPK activation leads to directactivation of specific G proteins, which remain complexed with ac-tivated NDPK (middle). We propose that glucose also activates isletendogenous histidine kinase, which we have shown to phosphorylatethe -subunit of trimeric G proteins (Fig. 2), thereby facilitating theactivation of cognate trimeric G protein. Glucose could also mediatehistidine phosphorylation of other proteins (e.g., ATP citrate lyase,aldolase, succinyl thiokinase) that are critical to glucose metabolism,thereby generating signals necessary for insulin secretion.

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munological methods, we localized - and -subunits ofATP- as well as GTP-sensitive isoforms of SCS in the-cell. In addition, using [-32P]ATP as a phosphoryldonor, we observed that the -subunit of SCS under-goes autophosphorylation at a histidine residue; copro- vision of exogenous succinate and CoA resulted inpronounced dephosphorylation of the phosphorylated-subunit of SCS. Taking these observations together,

we provide evidence for the localization of two distinctactivities of SCS in the -cell mitochondria. Whereas itis well established that ATP is critical for -cell metab-olism, we propose that GTP generated by the activa-tion of SCS, whose functional regulation is mediated via histidine phosphorylation, could promote keyfunctional roles in the mitochondrial metabolismthat lead to insulin secretion (36, 53).

As I review these cited studies, I think that the relayof high-energy phosphates as a consequence of proteinhistidine phosphorylation constitutes an importantnon-receptor-mediated activation of specific G proteins(and other proteins relevant to nutrient metabolism)by physiological stimuli such as glucose. Additional

studies are required to substantiate such a hypothesis.In this context, two recent studies have provided addi-tional support to our original formulation (52) for thenon-receptor-dependent activation of G proteins involving protein histidine phosphorylation and high-energy phosphate transfer. First, Cuello et al. (9)reported activation of trimeric G proteins by a high-energy phosphate transfer from the histidine-phospho-rylated NDPK to the -subunit of trimeric G proteins.Using bovine retinal and brain preparations, theseinvestigators observed that the B isoform of NDPKforms complexes with the -subunits of trimeric Gproteins and contributes to the activation of the respec-

tive G protein by increasing the high-energy phosphatetransfer from a transiently phosphorylated His266 inthe -subunit to the GDP bound to the -subunit, toyield an active conformation. In the second study,Hippe et al. (24) demonstrated the existence of a com-plex between NDPK (B isoform) and the -complex oftrimeric G proteins, and they implicated a role forNDPK in the phosphorylation of the -subunit, whichis then transferred to the GDP bound to the -subunit,resulting in its active, GTP-bound conformation. Inter-estingly, these findings are compatible with our recentobservations on the existence of NDPK and succinylthiokinase complexes in -cells (53), on the basis ofwhich we proposed a role for NDPK in the functional

regulation of succinyl thiokinase. It is likely that themitochondrial NDPK might interact with other mito-chondrial proteins as well. This is plausible, especiallyin light of recent observations of Srere and coworkers(87, 98) that clearly indicated the existence of com-plexes (appropriately termed metabolons) of sequen-tial metabolic enzymes involved in the tricarboxylicacid cycle. Together, it appears likely that the histidinekinase and various isoforms of NDPK that we charac-terized recently (Table 3) could subserve the function ofhistidine phosphorylation of key proteins (e.g., mono-meric G proteins or subunits of trimeric G proteins),

leading to the generation of appropriate signals neces-sary for physiological insulin secretion (37, 52, 53).

Several recent studies have identified additionalroles for NDPK, such as its ability to interact withguanine nucleotide exchange factors for specific G pro-teins and subserve the function of activating specificGTPases (77, 78, 111). Although these regulatorymechanisms have not been fully studied in the islet

-cell, we (95) and others (4) have obtained evidence toindicate localization of such factors (e.g., the guaninenucleotide exchange factor 1, or GRF1) in insulin-secreting cells. We (45) also described localization ofsimilar exchange factors in normal islet and clonal-cells, which appear to be regulated by phospholipase-derived mediators of insulin secretion (e.g., arachi-donic acid, lysophosphatidylcholine, and phosphatidicacid). In this context, we observed (unpublished obser-vations) potential regulation of the islet NDPK activityby lipid messengers of insulin secretion (e.g., arachi-donic acid). Although it seems likely, it remains to beseen whether nutrient-stimulated insulin secretion involves interplay between lipid messengers of insulinsecretion, NDPK, guanine nucleotide exchange factors,and their effector G proteins within confines of a stim-ulated -cell. Furthermore, studies by Wagner and Vu(101) have identified roles for NDPK in the phosphor-ylation of farnesyl and geranylgeranyl triphosphates,which form precursors for G protein isoprenylation. Inconclusion, a growing body of evidence is emerging tosuggest critical regulatory roles for this enzyme, whichwas originally believed to play the role of a house-keeping gene. On the basis of the above-mentionedreasons, it is logical to expect an increased interest inthe area of putative regulatory roles of protein histi-dine phosphorylation in metabolic function and stimu-

lus-secretion coupling, not only of the-cell but of otherendocrine cells as well.

ISLET G PROTEINS IN MODELS OF IMPAIRED

INSULIN SECRETION

Recent evidence from multiple laboratories appearsto suggest abnormalities in the expression and/or func-tion of G proteins in animal and in vitro models ofimpaired insulin secretion. The majority of these stud-ies were aimed at understanding the functional statusof trimeric as well as monomeric G proteins. A rela-tively large body of evidence is emerging on alterationsin the expression and function of G protein metabolism

in islets derived from the GK rat, a widely acceptedgenetically determined rodent model for human type 2diabetes. For example, we previously reported (67) thatinsulin secretion elicited in the presence of stimulatoryconcentrations of glucose, succinic acid methyl ester, ora depolarizing concentration of KCl was significantlyimpaired in GK rats. Interestingly, insulin secretionelicited by Mas was markedly increased above andbeyond the stimulatory effects of this compound incontrol Wistar rat islets. We also demonstrated a sig-nificant reduction in the ATP- as well as GTP-sensitivephosphorylation and catalytic function of NDPK in

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significant role in IL-1-mediated nitric oxide releasefrom isolated rat islets and clonal -cell preparations(47, 94, 95). Again, as above, specific inhibitors ofposttranslational modifications of G proteins, as wellas bacterial toxins, were utilized to decipher the role ofRas in this phenomenon. Compatible with these obser-vations are other reports that suggested key regulatoryroles for GTP in the survival of the islet -cell (see Ref.

65 for a review). Together, these data clearly providethe initial evidence, in the context of the -cell, thatGTP and G proteins play very important functionalroles in the normal functioning of the islet, and thatproapoptotic G proteins (e.g., Ras) play roles in thepropagation of cellular events responsible for the cyto-kine-induced loss of-cell mass, leading to the onset ofinsulin-dependent diabetes mellitus (47, 94, 95).Clearly, this area is in its infancy, and additionalstudies are needed to identify these candidate pro-andantiapoptotic G proteins. This is an important area ofinvestigation, since such data could provide valuableinsights into the development of therapeutic interven-tion modalities for the prevention of loss of-cell mass.

CONCLUSIONS AND FUTURE DIRECTIONS

From the discussion above, it is apparent that small-molecular-mass G proteins play key regulatory roles inthe stimulus-secretion coupling of the islet -cell.These conclusions were reached on the basis of studiesusing mostly biochemical, physiological, and limitedgene depletion approaches. We propose that glucose-mediated, receptor-independent activation of these Gproteins requires the intermediacy of protein histidinephosphorylation and subsequent relay of the high-en-ergy phosphate to GDP bound to G proteins to yieldtheir respective GTP-bound active conformation. It

also appears that alterations in the expression and/orfunctional activation of these proteins lead to impairedinsulin secretion. Furthermore, specific G proteins(e.g., Ras) seem to play proapoptotic roles in the islet-cell after exposure to cytokines. It will be necessaryto develop systems for the overexpression of G proteinsor application of antisense approaches for specific Gproteins (and their modifying enzymes), not only todeduce the physiological functions of these proteins inmodulating insulin secretion but also to develop poten-tial therapeutic approaches to states of perturbed met-abolic status and insulin release. For these reasons,there appears to be an immediate need for the devel-opment of novel inhibitors of G protein functions, es-pecially for those proteins that control and propagatesignal transduction steps leading to the generation ofnitric oxide, and consequently leading to the metabolicdysfunction and demise of the pancreatic -cell. Inaddition to these pharmacological probes, identifica-tion of candidate G proteins might help us in thedevelopment of novel bioengineered cell lines, whichare resistant to immune attack, for the treatment ofdiabetes in humans (14, 72).

I thank the Medical Research Service of the Department of Vet-erans Affairs for the Research Career Scientist Award. I sincerely

thank all of my former colleagues at the University of Wisconsin-Madison and my current associates at Wayne State University-Detroit who contributed to the work that I have described in thisreview.

DISCLOSURES

My research work was funded by the Department of VeteransAffairs (Merit Review and the Research Enhancement Award pro-gram grants), National Institute of Diabetes and Digestive and

Kidney Diseases (DK-56005), the American Diabetes Association,the Burroughs Wellcome Trust, and the Grodman Cure Foundation.

REFERENCES

1. Alex LA and Simon MI. Protein histidine kinases and signaltransduction in prokaryotes and eukaryotes. Trends Genet 10:133138, 1994.

2. Amin R, Chen HQ, Tannous M, Gibbs R, and Kowluru A.Inhibition of glucose- and calcium-induced insulin secretionfrom TC3 cells by novel inhibitors of protein isoprenylation.J Pharmacol Exp Ther 303: 8288, 2002.

3. Amin R, Chen HQ, Veluthakal R, Li J, Li G, and KowluruA. Novel roles for Rac1 in mastoparan-induced insulin secre-tion. Diabetes 52, Suppl 1: 1604, 2003.

4. Arava Y, Seger R, and Walker MD. GRF, a novel regulatorof calcium signaling, is expressed in pancreatic cells and

brain. J Biol Chem 274: 2444924452, 1999.5. Besant PG and Attwood PV. Detection of mammalian his-

tone H4 kinase that has yeast histidine kinase-like enzymaticactivity. Int J Biochem Cell Biol 32: 243253, 2000.

6. Birnbaumer L. Receptor-to-effector signaling through G pro-teins: roles for beta gamma dimers as well as alpha subunits.Cell 71: 10691072, 1992.

7. Boitard C, Larger E, Timsit J, Sempe P, and Bach JF.IDD: an islet or an immune disease? Diabetologia 37, Suppl 2:S90S98, 1994.

8. Crovello CS, Furie BC, and Furie B. Histidine phosphory-lation of P-selectin upon stimulation of human platelets: a novelpathway for activation-dependent signal transduction. Cell 82:279286, 1995.

9. Cuello F, Schulz RA, Heemeyer F, Meyer HE, Lutz S,Jakobs KH, Niroomand F, and Wieland T. Activation ofheterotrimeric G proteins by a high energy phosphate transfer

via nucleoside diphosphate kinase (NDPK) B and Gbeta sub-units. Complex formation of NDPK B with Gbeta gammadimers and phosphorylation of His-266 IN Gbeta. J Biol Chem278: 72207226, 2003.

10. Cunningham JM and Green IC. Cytokines, nitric oxide andinsulin secreting cells. Growth Regul 4: 173180, 1994.

11. Daniel S, Noda M, Cerione RA, and Sharp GW. A linkbetween Cdc42 and syntaxin is involved in mastoparan-stimu-lated insulin release. Biochemistry 41: 96639671, 2002.

12. Detimary P, Van den Berghe G, and Henquin JC. Concen-tration dependence and time course of the effects of glucose onadenine and guanine nucleotides in mouse pancreatic islets.J Biol Chem 271: 2055920565, 1996.

13. De Vos ML, Lawrence DS, and Smith CD. Cellular phar-macology of cerulenin analogs that inhibit protein palmitoyl-ation. Biochem Pharmacol 62: 985995, 2001.

14. Efrat S. Cell replacement therapy for type 1 diabetes. Trends Mol Med 8: 334340, 2002.

15. Feldman F and Butler LG. Detection and characterization ofthe phosphorylated form of microsomal glucose-6-phosphatase. Biochem Biophys Res Commun 36: 119125, 1969.

16. Frayon S, Pessah M, Giroix MH, Mercan D, Boissard C,Malaisse WJ, Portha B, and Garel JM. G.olf identificationby RT-PCR in purified normal pancreatic B cells and in isletsfrom rat models of non-insulin-dependent diabetes. BiochemBiophys Res Commun 254: 269272, 1999.

17. Gilman AG. G proteins: transducers of receptor-generatedsignals. Annu Rev Biochem 56: 615649, 1987.

18. Gonzalo S and Linder ME. SNAP-25 palmitoylation andplasma membrane targeting require a functional secretorypathway. Mol Biol Cell 9: 585597, 1998.

E681INVITED REVIEW



15/17

19. Hegde AN and Das MR. Ras proteins enhance the phosphor-ylation of a 38 kDa protein (P38) in liver plasma membrane.FEBS Lett 217: 7480, 1987.

20. Hertelendy ZI, Patel DG, and Knittel JJ. Pancreastatininhibits insulin secretion in RINm5F cells through obstructionof G-protein mediated, calcium-directed exocytosis. Cell Cal-cium 19: 125132, 1996.

21. Higashijima T, Burnier J, and Ross EM. Regulation of Giand Go by mastoparan, related amphiphilic peptides and hy-drophobic amines. J Biol Chem 265: 1417614181, 1990.

22. Higashijima T, Uzu S, Nakajima T, and Ross EM. Masto-paran, a peptide toxin from wasp venom, mimics receptors byactivating GTP-binding regulatory proteins (G-proteins). J BiolChem 263: 64916494, 1988.

23. Hillaire-Buys D, Mousli M, Landry Y, Bockaert J, Feh-rentsz JA, Carrette J, and Rouot B. Insulin releasing effectsof mastoparan and amphiphilic substance P receptor antago-nists on RINm5F insulinoma cells. Mol Cell Biochem 109:133138, 1992.

24. Hippe HJ, Lutz S, Curllo F, Knorr K, Vogt A, Jakobs KH,Wieland T, and Niroomand F.Activation of heterotrimeric Gproteins by a high energy phosphate transfer via nucleosidediphosphate kinase (NDPK) B and Gbeta subunits. Specificactivation of Gsalpha by an NDPK B Gbetagamma complex inH10 cells. J Biol Chem 278: 72277233, 2003.

25. Huang JM, Wei YF, Kim YH, Osterberg L, and Matthews

HR. Purification of a protein histidine kinase from the yeastSaccharomyces cerevisiae. The first member of this class ofprotein kinases. J Biol Chem 266: 90239031, 1991.

26. Jones PM, Mann FM, Persaud SJ, and Wheeler-Jones CP.Mastoparan stimulates insulin secretion from pancreatic beta-cells by effects at a late stage in the secretory pathway.Mol CellEndocrinol 94: 97103, 1993.

27. Jones PM and Persaud SJ. Protein kinases, protein phos-phorylation, and the regulation of insulin secretion from pan-creatic cells. Endocrine Rev 19: 429461, 1998.

28. Kim Y, Huang J, Cohen P, and Matthews H. Protein phos-phatases 1, 2A, and 2C are protein histidine phosphatases.J Biol Chem 268: 1851318518, 1993.

29. Kimura N, Shimada N, Fukuda M, Ishijima Y, MiyazakiH, Ishii A, Takagi Y, and Ishikawa N. Regulation of cellularfunctions by nucleoside diphosphate kinases in mammals. J Bioenerg Biomemb 32: 309315, 2000.

30. Klumpp S and Krieglstein J. Phosphorylation and dephos-phorylation of histidine residues in proteins. Eur J Biochem269: 10671071, 2002.

31. Komatsu M, Aizawa T, Yokokawa N, Sato Y, Okada N,Takasu N, and Yamada T. Mastoparan-induced hormonerelease from rat pancreatic islets. Endocrinology 130: 221228,1992.

32. Komatsu M, McDermott AM, Gillison SL, and Sharp GW.Mastoparan stimulates exocytosis at a Ca (2)-independentlate site in stimulus-secretion coupling. Studies with theRINm5F beta-cell line. J Biol Chem 268: 2329723306, 1993.

33. Komatsu M, Noda M, and Sharp GW. Nutrient augmenta-tion of calcium-dependent and calcium-independent pathwaysin stimulus-coupling to insulin secretion can be distinguishedby their guanosine triphosphate requirements: studies on ratpancreatic islets. Endocrinology 139: 11721183, 1998.

34. Konrad RJ, Young RA, Record RD, Smith RM, ButkeraitP, Manning D, Jarett L, and Wolf BA. The heterotrimericG-protein Gi is localized to the insulin secretory granules ofbeta-cells and is involved in insulin exocytosis. J Biol Chem270: 1286912876, 1995.

35. Kowluru A. Evidence for the carboxyl methylation of nuclearlamin-B in the pancreatic cell.Biochem Biophys Res Commun268: 249254, 2000.

36. Kowluru A. Adenine and guanine nucleotide-specific succinyl-CoA synthetases in the clonal beta-cell mitochondria: implica-tions in the beta-cell high-energy phosphate metabolism inrelation to physiological insulin secretion. Diabetologia 44: 8994, 2001.

37. Kowluru A. Identification and characterization of a novelprotein histidine kinase in the islet cell: evidence for its

regulation by mastoparan, an activator of G-proteins and insu-lin secretion. Biochem Pharmacol 63: 20912100, 2002.

38. Kowluru A. Defective protein histidine phosphorylation inislets from the Goto-Kakizaki diabetic rat. Am J Physiol Endo-crinol Metab 285: E498E503, 2003.

39. Kowluru A and Amin R. Inhibitors of posttranslational mod-ifications of G-proteins as probes to study the pancreatic cellfunction: potential therapeutic implications. Curr Drug Targets Immune Endocr Metabol Disord 2: 129139, 2002.

40. Kowluru A, Chen HQ, and Tannous M. Novel roles for Rho

subfamily of GTP-binding proteins in succinate-induced insulinsecretion from TC3 cells. Endocr Res 2003. In press.

41. Kowluru A, Li G, Rabaglia ME, Segu VB, Hofmann F, Aktories K, and Metz SA. Evidence for differential roles ofthe Rho subfamily of GTP-binding proteins in glucose- andcalcium-induced insulin secretion from pancreatic beta cells. Biochem Pharmacol 54: 10971108, 1997.

42. Kowluru A, Li G, and Metz SA. Glucose activates the car-boxyl methylation of subunits of trimeric GTP-binding pro-teins in pancreatic cells. J Clin Invest 100: 596610, 1997.

43. Kowluru A and Metz SA. Characterization of nucleosidediphosphokinase activity in human and rodent pancreatic cells: evidence for its role in the formation of guanosine triphos-phate, a permissive factor for nutrient-induced insulin secre-tion. Biochemistry 33: 1249512503, 1994.

44. Kowluru A and Metz SA. GTP and its binding proteins in the

regulation of insulin exocytosis. In: Molecular Biology of Dia-betes, edited by Draznin B and LeRoith D. Totowa, NJ: Hu-mana, 1994, p. 249283.

45. Kowluru A and Metz SA. Regulation of guanine-nucleotidebinding proteins in islet subcellular fractions by phospholipase-derived lipid mediators of insulin secretion. Biochim BiophysActa 1222: 360368, 1994.

46. Kowluru A and Metz SA. Stimulation by prostaglandin E2 ofa high-affinity GTPase in the secretory granules of normal ratand human pancreatic islets. Biochem J 297: 399406, 1994.

47. Kowluru A and Morgan NG. GTP-binding proteins in cellsurvival and demise: the emerging picture in the pancreatic cell. Biochem Phramacol 63: 10271035, 2002.

48. Kowluru A, Robertson RP, and Metz SA. GTP-bindingproteins in the regulation of pancreatic cell function. In:Diabetes Mellitus. A Fundamental and Clinical Text, edited byLeRoith D, Taylor SI, and Olefsky JM. Philadelphia, PA: Lip-pincott Williams & Wilkins, 2000, p. 7894.

49. Kowluru A, Seavey SE, Li G, Sorenson RL, Weinhaus AJ,Nesher R, Rabaglia ME, Vadakekalam J, and Metz SA.Glucose- and GTP-dependent stimulation of the carboxyl meth-ylation of Cdc42 in rodent and human pancreatic islets andpure cells. J Clin Invest 98: 540555, 1996.

50. Kowluru A, Seavey SE, Rabaglia ME, and Metz SA. Non-specific stimulatory effects of mastoparan on pancreatic isletnucleoside diphosphate kinase activity: dissociation from insu-lin secretion. Biochem Pharmacol 49: 263266, 1995.

51. Kowluru A, Seavey SE, Rabaglia ME, Nesher R, and MetzSA. Carboxyl methylation of the catalytic subunit of proteinphosphatase 2A in insulin-secreting cells: evidence for func-tional consequences on enzyme activity and insulin secretion.Endocrinology 137: 23152323, 1996.

52. Kowluru A, Seavey SE, Rhodes CJ, and Metz SA. A novel

regulatory mechanism for trimeric GTP-binding proteins in themembrane and secretory granule fractions of human and ro-dent beta cells. Biochem J 313: 97107, 1996.

53. Kowluru A, Tannous M, and Chen HQ. Localization andcharacterization of the mitochiondrial isoform of the nucleosidediphosphate kinase in the pancreatic cell: evidence for itscomplexation with mitochondrial succinyl-CoA synthetase. Arch Biochem Biophys 398: 160169, 2002.

54. Leiser M, Efrat S, and Fleischer N. Evidence that Rap1carboxyl-methylation is involved in regulated insulin secretion.Endocrinology 136: 25212530, 1995.

55. Li G, Kowluru A, and Metz SA. Characterization of prenyl-cysteine methyltransferase in insulin-secreting cells. BiochemJ 316: 345351, 1996.

E682 INVITED REVIEW



16/17

56. Li G, Regazzi R, Roche E, and Wollheim CB. Blockade ofmevalonate roduction by lovastatin attenuates bombesin and

vasopressin potentiation of nutrient-induced insulin secretionfrom HIT-T15 cells. Biochem J 289: 379385, 1993.

57. Li J, Luo R, Kowluru A, and Li G. Involvement of Rac1, asmall G-protein, in islet cell growth and insulin secretion.Diabetes 52, Suppl 1: 1616, 2003.

58. Linder ME and Deschenes RJ. New insights into the mech-anisms of protein palmitoylation. Biochemistry 42: 43114320,2003.

59. MacDonald MJ. Elusive proximal signals of beta-cells forinsulin secretion. Diabetes 39: 14611466, 1990.

60. Malaisse WJ. Hormonal and environmental modification ofislet activity. In: Handbook of Physiology. Endocrine Pancreas.Bethesda, MD: Am. Physiol. Soc., 1972, vol. I, sect. 7, chapt. 14,p. 237260.

61. Mandrup-Poulsen T. The role of interleukin-1 in the patho-genesis of IDDM. Diabetologia 39: 10051029, 1996.

62. Matthews HR. Protein kinases and phosphatases that act onhistidine, lysine, or arginine residues in eukaryotic proteins: apossible regulator of the mitogen activated protein kinase cas-cade. Pharmocol Ther 67: 323350, 1995.

63. Matthews HR and Chan K. Protein histidine kinase. MethMol Biol 124: 171182, 2001.

64. McDaniel ML, Kwon G, Hill JR, Marshall CA, and CorbettJA. Cytokines and nitric oxide in islet inflammation and dia-

betes. Proc Soc Exp Biol Med 11: 2432, 1996.65. Metz SA and Kowluru A. Inosine monophosphate dehydro-genase: a molecular switch integrating pleiotropic GTP-depen-dent cell functions. Proc Assoc Am Physicians 111: 335346,1999.

66. Metz SA, Meredith M, Rabaglia ME, and Kowluru A.Small elevations of glucose concentration redirect and amplifythe synthesis of guanosine 5-triphosphate in rat islets. J ClinInvest 92: 872882, 1993.

67. Metz SA, Meredith M, Vadakekalam J, Rabaglia ME, andKowluru A. A defect late in stimulus-secretion coupling im-pairs insulin secretion in Goto-Kakizaki diabetic rats. Diabetes48: 17541762, 1999.

68. Metz SA, Rabaglia ME, and Pintar TJ. Selective inhibitorsof GTP-synthesis impede exocytotic insulin release from intactrat islets. J Biol Chem 267: 1251712527, 1992.

69. Metz SA, Rabaglia ME, Stock JB, and Kowluru A. Modu-

lation of insulin secretion from normal rat islets by inhibitors ofthe posttranslational modifications of GTP-binding proteins. Biochem J295: 3140, 1993.

70. Motojima K and Goto S. Histidyl phosphorylation and de-phosphorylation of P36 in liver extracts. J Biol Chem 269:90309037, 1994.

71. Nederkoorn PHJ, Timmerman H, Timms D, Wilkinson AJ, Kelly DR, Broadley KJ, and Davies RH. Stepwisephosphorylation mechanisms and signal transmission within aligand-receptor-G-protein complex. J Mol Struct 452: 2547, 1998.

72. Newgard CB, Clark S, BeltrandelRio H, Hohmeier HE,Quaade C, and Normington K. Engineered cell lines forinsulin replacement in diabetes: current status and futureprospects. Diabetologia 40, Suppl 2: S42S47, 1997.

73. Newgard CB and McGarry JD. Metabolic coupling factors inpancreatic cell signal transduction. Ann Rev Biochem 64:689719, 1995.

74. Ohara-Imaizumi M, Nakamichi Y, Ozawa S, Katsuta H,Ishida H, and Nagamatsu S. Mastoparan stimulates GABArelease from MIN6 cells: relationship between SNARE proteinsand mastoparan action. Biochem Biophys Res Commun 289:10251030, 2001.

75. Ohtsuki K, Ikeuchi T, and Yokoyama M. Characterizationof nucleoside-diphosphate kinase-associated guanine nucleo-tide-binding proteins from HeLaS3 cells. Biochim Biophys Acta882: 322330, 1986.

76. Ostenson CG, Zaitsev S, Berggren PO, Efendic S, LangelU, and Bartfai T. Galparan: a powerful insulin-releasing chi-meric peptide acting at a novel site. Endocrinology 138: 330833013, 1997.

77. Otsuki Y, Tanaka M, Yoshi S, Kawazoe N, Nakaya K, andSigimura H. Tumor metastasis suppressor nm23H1 regulatesRac1 GTPase by interaction with Tiam1. Proc Natl Acad SciUSA 98: 43854390, 2001.

78. Palacios F, Schweitzer JK, Boshans RL, and DSouza-Schorey C. ARF6-GTP recruits Nm23-H1 to facilitate dy-namin-mediated endocytosis during adherens junctions disas-sembly. Nat Cell Biol 4: 929936, 2002.

79. Piacentini L and Niroomand F. Phosphotransfer reactionsas a means of G protein activation. Mol Cell Biochem 157:

5963, 1996.80. Portela-Gomes GM and Abdel-Halim SM. Overexpression

of Gs proteins and adenylyl cyclase in normal and diabeticislets. Pancreas 25: 176181, 2002.

81. Prentki M and Matschinsky FM. Calcium, cAMP, and phos-pholipid-derived messengers in coupling mechanisms of insulinsecretion. Physiol Rev 67: 12231226, 1987.

82. Rabuzzo AM, Buscema M, Caltabiano V, Anello M, De-gano C, Patane G, Vigneri R, and Purrello F. Interleukin1 inhibition of insulin release in rat pancreatic islets: possibleinvolvement of G-proteins in the signal transduction pathway.Diabetologia 38: 779784, 1995.

83. Regazzi R, Kikuchi A, Takai Y, and Wollheim CB. Thesmall GTP-binding proteins in the cytosol of insulin-secretingcells are complexed to GDP dissociation inhibitor proteins.J Biol Chem 267: 1751217519, 1992.

84. Regazzi R, Sasaki T, Takahashi K, Jonas JC, Volker C,Stock JB, Takai Y, and Wollheim CB. Prenylcysteine ana-logs mimicking the C-terminus of GTP-binding proteins stim-ulate exocytosis from permeabilized HIT-T15 cells: comparisonwith the effects of Rab3AL peptide.Biochim Biophys Acta 1268:269278, 1995.

85. Robertson RP, Seaquist ER, and Walseth TF. G-proteinsand modulation of insulin secretion. Diabetes 40: 16, 1991.

86. Schmidt M, Rumenapp U, Bienek C, Keller J, von Eichel-Streiber C., and Jakobs KH. Inhibition of receptor signalingto phospholipase D by Clostridium difficile toxin B. Role of Rhoproteins. J Biol Chem 271: 24222466, 1996.

87. Srere P. Complexities of metabolic regulation. Trends BiochemSci 19: 519520, 1994.

88. Srivastava M, Atwater I, Glasman M, Leighton X, GopingG, Caohuy H, Miller G, Pichel J, Westphal H, Mears D,Rojas E, and Pollard HB. Defects in inositol 1,4,5-triphos-

phate receptor expression, Ca2

signaling, and insulin secre-tion in the anx 7(/-) knockout mouse. Proc Natl Acad Sci USA96: 1378313788, 1999.

89. Steeg PS, Palmieri D, Ouatas T, and Salerno M. Histidinekinases and histidine phosphorylated proteins in mammaliancell biology, signal transduction and cancer. Cancer Lett 190:112, 2003.

90. Straub SG, James RF, Dunne MJ, and Sharp GW. Glucoseaugmentation of mastoparan-stimulated insulin secretion inrat and human pancreatic islets. Diabetes 47: 10531057, 1998.

91. Straub SG, Yajima H, Komatsu M, Aizawa T, and SharpGWG. The effects of cerulenin, an inhibitor of protein acylation,on the two phases of glucose-stimulated insulin secretion. Dia-betes, Suppl 1: S91S95, 2002.

92. Takai Y, Sasaki T, and Matozaki T. Small GTP-bindingproteins. Physiol Rev 81: 153208, 2001.

93. Tan E, Besant PG, and Attwood PV. Mammalian histidinekinases: do they REALLY exist? Biochemistry 41: 38433851,2002.

94. Tannous M, Amin R, Popoff MR, Fiorentini C, andKowluru A. Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonalbeta (HIT-T15) cells. Biochem Pharmacol 62: 14591468, 2001.

95. Tannous M, Veluthakal R, Amin R, and Kowluru A. IL-1-induced nitric oxide release

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Lessons From Models of Impaired Insulin Secretion