THE JOURNU. OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 35, Issue of December 15, pp. 21590-21595,199O Printed in U.S.A.

An Aspartate Conserved among G-protein Receptors Confers Allosteric Regulation of cw2-Adrenergic Receptors by Sodium*

(Received for publication, August 1, 1990)

Debra A. HorstmanS, Suzanne Brandon, Amy L. Wilsonj, Cheryl A. Guyer, Edward J. Cragoe, Jr.Y, and Lee E. Limbird From the Department of Pharmacology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6600 and lBox 631548, Nacogdoches, Texas 75963

The residue involved in sodium regulation of G-pro- tein-coupled receptors has been identified by site-di- rected mutagenesis of the az-adrenergic receptor gene. Mutation of Asp-79 to Asn-79 entirely eliminates al- losteric regulation of ligand binding by monovalent cations without perturbing the selectivity of adrener- gic binding or allosteric modulation of that binding by amiloride analogs. The high degree of conservation of this aspartate residue in all G-protein-coupled recep- tors, without even a conservative change to glutamate, underscores the probable importance of this allosteric regulation.

Ligand binding to many members of the G-protein-coupled family of receptors is modulated allosterically by monovalent cations. Prominent among this group are receptors linked to the inhibition of adenylyl cyclase such as az-adrenergic (l-5), Dz-dopaminergic (6), p and 6 opiate (7-9), somatostatin (lo), and certain subtypes of muscarinic (11, 12) receptors. Mono- valent cation modulation of binding also has been demon- strated for @-adrenergic receptors (13), which are linked to stimulation of adenylyl cyclase. In all of these systems, mon- ovalent cations (Na+ z Li’ > K’) have reciprocal effects on agonist versus antagonist interaction at the receptors, thus resembling guanine nucleotide regulation of receptor-ligand interactions at G-protein-coupled receptors (14). However, several lines of evidence indicate that the effect of monovalent cations i$ not mediated via G-proteins (15), including the observation that these allosteric effects are retained in ho- mogeneous preparations of the porcine brain a*-receptor (3).

Recent studies (16) in which the purified porcine brain (YZ- adrenergic receptor was treated with trypsin to generate a “hydrophobic tryptic core” of the receptor (see schematic, Fig. 2), indicate that allosteric modulation of this core receptor by monovalent cations is quantitatively indistinguishable from that of the native receptor. Since we anticipated that a Na+- binding pocket might possess a carboxylate residue as a coun- ter ion, the finding that Na+ modulation is retained by the hydrophobic tryptic core significantly refined our ability to propose potential loci for Na’ binding since few aspartate or glutamate residues are retained in the core receptor. Because changes in receptor-agonist interactions correlate with changes in intracellular Na+ concentration (17), exofacial Asp

*This research was supported in part by National Institutes of Health Grants HL25182 and HL43671. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by Training Grant GM07628.. $ Recipient of a Fellowship Award from the AHA.

and Glu residues could be excluded, at least initially, from consideration as potential sites of Na’ interaction with the receptor. Also, since Asp-113 has been implicated as a residue essential for catecholamine binding at adrenergic receptors (l&33), we postulated that it likely would not also be involved in allosteric modulation of that binding. Consequently, those carboxylate residues (such as Asp-79, Asp-130, and Asp-432) that are accessible from the cytoplasmic compartment and are not implicated in adrenergic ligand binding were mutated to asparagine residues using oligonucleotide-directed muta- genesis of the gene coding for the porcine cypA-adrenergic receptor (19). All mutant cYz-receptors examined bound the alp-receptor antagonist, [3H]yohimbine, and demonstrated similar rank order of potencies for both agonists and antago- nists in competing for this binding as observed for the wild type cY2-adrenergic receptor. However, the present study dem- onstrates that the mutation of aspartate 79 to asparagine 79 resulted in complete loss of the ability of Na+ to modulate (YY receptor-ligand interactions.

MATERIALS AND METHODS

Materials-[r-32P]ATP (6000 Ci/mmol) and [3H]yohimbine (70- 90 Ci/mmol) were purchased from Du Pont-New England Nuclear. The a*-adrenergic receptor photolabel was synthesized according to the method of Lanier et al. (24). Amiloride analogs were purchased from Dr. Edward J. Cragoe, Jr. Digitonin was purchased from Gallard- Schlesineer Chemical Manufacturing Cornoration, Carle Place, NY. Restrict& endonucleases were obtained e&her from Promega Biotec (Madison, WI), New England Biolabs, or Bethesda Research Labo- ratories. Suppliers for reagents used in protocols involving recombi- nant DNA methodologies are indicated below.

Oligonucleotide-directed Mutagenesis-The gene encoding the por- cine cYB-adrenergic receptor that had been inserted into the polylinker region of m13mp18 at EcoRI/XbaI sites was used as the single- stranded template. Oligo-directed mutagenesis was performed accord- ing to Zoller and Smith (20). Identification of plaques containing DNA of mutant sequence was performed using radiolabeled mutant olizonucleotides as nrobes (21). Single-stranded DNA was isolated frok the positive &aques and the-presence of the mutation was verified by DNA sequencing (Sequenase kit, U. S. Biochemical Corp.). Double-stranded templates were synthesized by annealing ml3 uni- versal primer (U. S. Biochemical Corp.) with the mutant templates and then extending the templates using deoxynucleotides and Klenow fragment of DNA polymerase (Promega Biotec). DNA cassettes con- taining the mutated sequence that encoded for Asn-79, Asn-130, or Asn-432 were obtained by treating the double-stranded templates with the appropriate restriction endonucleases. These inserts encod- ine mutant seouences were subcloned into the expression vector, peMV4 a,AR (19). The pCMV4 expression vector was synthesized and generouslv nrovided bv Dr. David W. Russell (University of Texai Health sciences Center, Dallas). The construction of the vector is described by Anderson et al. (22). Double-stranded sequencing was performed to verify that the correct mutant sequence was present in each expression plasmid and that the mutant insert was ligated into the vector without error.

Growth of COSM6 Cells and Transfection with Wild Type and

21590

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

al-Receptor Mutant Lacks Allosteric Effects of Na’ 21591

71 kDa& -

Yoh

MW A~432 Marker

- 92.5

- 66.1

- 45

-+-+-+-+ FIG. 1. Photoaffinity labeling of porcine a2-adrenergic

receptors expressed transiently in COSMG. aa-Receptors ex- pressed in COSMG cells were partially purified from digitonin-solu- bilized preparations by WGA-agarose chromatography as described under “Materials and Methods.” Samples containing approximately 0.1 pmol of az-receptor were photolabeled with 2 nM ‘*‘I-rau-AzPec in the absence or presence of 1 pM yohimbine as described under “Materials and Methods.”

TABLE I Specificity of agonists and antagonists in interacting

with wild type and mutant ol+eceptors The data shown represent the EC& values + SE. obtained from

three independent experiments performed in duplicate. The order of potency of agonists (oxymetazoline > UK14304 > epinephrine) and the relative ineffectiveness of prazosin in competing for [RH]yohim- bine binding is characteristic of an a*-receptor of the agr\-subtype (45).

Agonists

&-Receptor

Wild type Asp-79 + Asn-79 Asp-130 + Asn-130 Asp-432 + Asn-432

Oxymetszoline UK14304 Epinephrine

ItM PM PM 35.0 + 5 0.41 + 0.05 3.8 + 0.17 75.0 + 15 1.8 I 0.25 8.7 + 0.67 28.0 + 5 0.48 f 0.01 7.2 + 0.44 73.3 f 9 0.36 f 0.02 2.3 f 0.37 Antagonists

as-Receptor

Wild type Asp-79 + Asn-79 Asp-130 + Asn-130 Asp-432 + Asn-432

Yohimbine

RM

10 + 0.9 43 + 2.5

19.0 + 1.0 8.7 + 0.9

Idazoxan

IlM 23 f 6.5

180 f 0 33 f 2.5 16 + 3.5

Prazosin

nhf 10,000

>10,000 10,000 10,000

Mutant Expression Plasm&-COSMG cells are a subclone of COS-7 cells and were obtained from Edith Womack in the laboratory of J. L. Goldstein (University of Texas Southwestern Medical Center, Dallas). COSMG cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin/ streptomycin as described (19). Expression of the as-receptor gene was accomplished by transient transfection of COSMG cells using a DEAE-dextran procedure (19, 23). At 72-h posttransfection, mem- branes were made by hypotonic lysis of the cells (19). Expression of cur-adrenergic receptors was measured in the cell membranes or in digitonin-solubilized preparations of the membranes by incubation with [3H]yohimbine.

Solubilization of as-Adrenergic Receptors and Ligand Binding As- says-Membranes, either fresh or from stocks stored at -70 “C, were collected by centrifugation at 39,000 x g for 30 min at 4 “C. The pellet was homogenized in buffer containing 1% digitonin, 20 mM HEPES,’ 25 mM glycylglycine, 100 mM NaCl, and 5 mM EGTA by 10 up-and-down strokes in a motor-driven teflon glass homogenizer. The sample was sonicated in an ice bath for 30 min and then centrifuged at 100,000 x g for 60 min at 4 “C (19). Except for the Asn-79 mutant, recovery of a,-receptor binding was about 50% as estimated by [3H]yohimbine binding of the solubilized preparation as compared to the membrane preparation. For the Asn-79 mutant, the apparent yield of solubilized a*-receptor was 5-lo-fold lower than for

’ The abbreviations used are: HEPES, 4-(2-hydroxyethyl)-l-piper- azineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)] tetraacetic acid; NMDG-Cl, N-methyl-D-glucamine chloride; WGA, wheat germ agglutinin; SDS, sodium dodecyl sulfate; I?-rau-AzPec,l 7~-hydroxy-20a-yohimban-l6~[~-(4-azido-3-[’25I]iodo)penethyl] carboxyamide (24); EC&,, concentration of competitor that effectively competes for 50% of radioligand binding.

wild type receptor. This apparent reduced yield may be due, at least in part, to the observation that the intrinsic rate of [“Hlyohimbine dissociation from this mutant was faster than for wild type or Asn- 130 and Asn-432 mutants, thus decreasing the fraction of a?-receptors trapped as the “bound” fraction following G-50 chromatography.

In order that solubilized receptor preparations could be assayed in low (40 mM) NaCl-containing solutions, solubilized az-adrenergic receptor preparations were passed over Sephadex G-50 columns, and aa-adrenergic receptors were eluted in the column void volume with Na+ free buffer (19) containing 0.2% digitonin, 25 mM glycylglycine, 40 mM HEPES, 100 mM NMDG-Cl, and 5 mM EGTA. The choice of 40 mM Na’ derives from earlier findings in human platelet, porcine brain cortex, and COSMG cells in which the allosteric effects of Na+ are maximal at 40 mM. The Na’ substitute, NMDG-Cl, was added to control incubations to maintain a constant ionic strength (3).

In experiments in which the rate of [nH]yohimbine dissociation from the a*-receptor was measured, the solubilized preparation was first incubated with 7.5 nM [3H]yohimbine for 60 min at 15 “C. The incubation temperature was then reduced to 10 “C for 30 min, since the rate of [3H]yohimbine dissociation was to be monitored at 10 “C. Prior to initiating the dissociation phase, specific binding of [“HI yohimbine to an aliquot of the incubation was determined and defined as binding at t = 0 of the dissociation phase. To initiate the dissocia- tion phase, unlabeled yohimbine was added to a final concentration of 50 PM; 5 min later the potential allosteric modifier (Na+ or amiloride analog) was added (19). Separation of bound from free radioligand was performed using 4-ml Sephadex G-50 columns as described previously (3, 19).

For competition binding experiments with agonists and antago- nists, digitonin-solubilized a*-receptor preparations were incubated with 7.5 nM [3H]yohimbine for 90 min at 15 “C, and incubations were terminated by chromatography through Sephadex G-50 columns. Agonist potency was evaluated in the absence of Na’ (40 mM NMDG- Cl present as Na’ substitute) whereas antagonist potency was eval- uated in the presence of 40 mM NaCl. Data are presented as E&Q values rather than KI values. Typically only 6-8 concentrations of competitor were used per competition binding profile to estimate the EC50 of the competitor in inhibiting [3H]yohimbine binding, and more detailed curves would be required to rigorously establish whether or not the competition profiles were of so-called “normal” steepness, as predicted for reversible bimolecular reactions occurring between li- gand and receptor via mass action law. In addition, the G-50 chro- matography procedure for resolving bound from free radioligand takes 14-30 min, depending on the “age” of the G-50 columns, and thus the equilibrium between bound and free radioligand is likely perturbed during this interval. Consequently, all of the assumptions required for applying the Cheng-Prusoff equation, EC& = Kr (1 + [ radioligand] /KDradioligsnd), for calculating K, values for competitors from EC50 estimates are not necessarily met in our studies. It should be noted, however, that the affinity of the mutant and wild type receptors for yohimbine is internally consistent when determined using saturation binding strategies (Fig. 2) or from competition binding studies (see Table I). Thus, if we were to assume that the Cheng-Prusoff equation could be applied to the competition data, then the K, for yohimbine from the competition binding studies for the wild type receptor assayed in the presence of Na+ would be calculated to be 3.4 nM, which compares well with the KO for [3H]yohimbine estimated from the saturation binding studies shown in Fig. 2e, 3.9 nM.

Saturation binding studies in the absence (40 mM NMDG-Cl) or presence of 40 mM NaCl were performed by incubating detergent- solubilized as-receptors with increasing concentrations of [3H]yohim- bine for 90 min at 15 “C and terminated by Sephadex G-50 chroma- tography (3, 19).

WGA-At?arose ChromatoaraDhv of Dtiitonin-solubilized Prepara- tions of CO%M6 Cells Tran&ctkd”witk Wk Type and Mutant Eipres- sion Plasm&-Digitonin-solubilized preparations of wild type and mutant a*-receptors containing the equivalent of 1 X lo6 cpm of binding were batch-adsorbed onto wheat germ agglutinin-agarose by overnight rotation with the resin. The WGA-agarose was equilibrated in 0.2% digitonin, 25 mM glycylglycine, 100 mM NaCl, and 5 mM EGTA in the presence of 10 mM MgCl I al-receptors were occupied by the antagonist phentolamine during WGA-agarose chromatogra- phy by maintaining phentolamine at 10 pM concentrations during the overnight adsorption phase. The resin was then washed, packed, and eluates tested for a*-receptor binding activity as described pre- viously (19).

Photoaffinity Labeling of Expressed Wild Type and Mutant a?- Adrenergic Receptors-olz-Receptor preparations purified by WGA

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

21592 wReceptor Mutant Lacks Allosteric Effects of Na+

0 -acidic residues removed by tryptic cleavage Extracellular

l A n -acidic residues remaining In hydrophobic tryptlc core

n - exofaclal A - inVOlVed in adrenergic ligand binding

0 -accessible from cytoplasm, possible loci for allosteric modulalioti by mohovalent cations

WILD TYPE A-,$.)+ tm+l7g ASP130+ ASN130

ALLOSTERIC EFFECTS OF SODIUM

3 It-yohlmblne Added, pmol

ALLOSTERIC EFFECTS OF AMILORIDE ANALOGS

il’~~~~~ 0 30 60 60 30 60 60 36 60 60 30 60 60

Time, Minutes

FIG. 2. Allosteric modulation of wild type and mutant az-adrenergic receptors. Upperpanel, a schematic diagram of the a*-receptor illustrates the predicted membrane topography of the receptor. Tryptic cleavage of the a*-receptor generates a “hydrophobic tryptic core” (represented by a thick line) that retains adrenergic binding as well as allosteric modulation of that binding by Na+ and amiloride analogs (16). Lower panel, (3H]yohimbine dissociation from digiton-solubilized receptor preparations derived from COSMG cells transiently expressing wild type and mutant a,-receptor genes was monitored as described under “Materials and Methods.” a-d, effect of 40 mM NaCl on the rate of dissociation of [3H]yohimbine from solubilized wild type (a), or Asn-79 (b), Asn-130 (c), and Asn-432 (d) mutant al-adrenergic receptors. The effects of 40 mM Na+ on saturation binding of [3H]yohimbine to digitonin-solubilized preparations of wild type (e), Asn-79 (fi, Asn-130 (g), and Asn-432 (h) mutant a,-receptors are illustrated, The K. values given were determined by Scatchard (Rosenthal) analysis of the saturation binding data. i-1, effects of amiloride analogs of the rate of [3H]yohimbine dissociation from solubilized wild type (i), Asn- 79 (i), Asn-130 (k), and Asn-432 (I) az-receptors. Control incubations contained 100 pM dimethylformamide, the diluent for ethylisopropylamiloride (EIA) and chlorobenzyldimethylbenzamil (CBDMB). EIA = 100 PM; CBDMB = 100 GM. For all dissociation data, the values given are mean + S.E. for at least three independent experiments performed in triplicate.

chromatography were incubated with 2 nM “‘1-rau-AzPec in 0.2% RESULTS AND DISCUSSION digitonin, 25 mM glycylglycine, 100 mM NaCl, 5 mM EGTA, and 400 mM N-acetylglucosamine (pH 7.6) for 3 h at 15 “C in the dark in the a,-Adrenergic receptor binding in membrane preparations absence (total binding) or presence (to define nonspecific binding) of from transiently transfected COSMG cells (19) was identified 1 pM yohimbine. Just prior to photolysis, 1 mM glutathione was added to scavenge any reactive intermediates present. Photolysis was per-

using the antagonist [3H]yohimbine and was comparable

formed as described (16, 19, 24) in quartz tubes by irradiation at 300 (fmoles/mg of membrane protein) for wild type and mutant

nm for 3 min at 4 “C. Samples were quenched by the addition of receptors (data not shown). The wild type and mutant CYZ- SDS-sample buffer and subjected to SDS-polyacrylamide gel electro- receptor proteins demonstrate similar apparent molecular phoresis and autoradiography (16, 19). masses (approximately 71 kDa) when identified by the 012-

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

oc2-Receptor Mutant Lacks Allosteric Effects of Na+ 21593

60

0 lo* 10’5 10

Loglo [Epinephrine]

FIG. 3. Competition of (-)epinephrine with [3H]yohimbine for binding to wild type and Asn-79 a2-receptors. Digitonin- solubilized receptor preparations were incubated with 7.5 nM [3H] yohimbine and increasing concentrations of (-)epinephrine in the absence (40 mM NMDG-Cl) or presence of 40 mM NaCl as described previously (19). The data shown (mean f S.E.) are the average of at least three experiments performed in duplicate. The shift in the position of the curve for epinephrine competition for [3H]yohimbine binding also was evaluated for the Asn-130 mutant (lo-fold) and for the Asn-432 mutant (3-fold) as compared with the shift in the wild type (7-g-fold), when competition was assessed in the absence UWW.S presence of 40 mM Na+.

adrenergic photoaffinity label, ‘251-rau-AzPec (24) and ana- lyzed by SDS-polyacrylamide gel electrophoresis (CL Fig. 1). To further investigate the functional integrity of the mutant a*-receptors, competition of agonists or antagonists with [3H] yohimbine for binding to digitonin-solubilized preparations of wild type and mutant a*-adrenergic receptor was performed as described under “Materials and Methods.” The order of potency of structurally diverse cuz-agonists and antagonists for binding to the a*-receptor was similar for the Asn-79, Asn- 130, and Asn-432 mutant proteins and the wild type o(~- receptor (cf. Table I). The EC,,-, values for the antagonists, yohimbine and idazoxan, were slightly greater for the Asn-79 mutant receptors as compared to the wild type receptor.

Since the mutant cuz-receptors appeared to possess appro- priate structural and adrenergic binding properties, we eval- uated allosteric modulation of the receptor.’ Allosteric mod-

* Digitonin-solubilized (3) rather than membrane preparations of wild type and mutant ol,-receptors were used to measure [3H]yohim- bine dissociation rates in order to circumvent potential variability arising from the nonuniform distribution of receptors in COSMG membrane preparations that is anticipated following transient trans- fection of the cells (19).

ulation of az-adrenergic receptor-ligand interactions is iden- tified most easily by monitoring dissociation rates of adrenergic ligands from the receptor (2, 3). As illustrated in Fig. 2a, Na+ increases the rate of [3H]yohimbine dissociation from the solubilized wild type receptor. Since the adrenergic ligand binding site is occupied by either tritiated or unlabeled yohimbine during the dissociation phase, the effects of Na’ were interpreted to be mediated via interaction at another, i.e. “allosteric,” site. The rate of [3H]yohimbine dissociation from mutant receptors Asn-130 (Fig. 2~) and Asn-432 (Fig. 2d) is indistinguishable from that for the wild type receptor, as is the ability of Na’ to accelerate this dissociation rate. In contrast, the Asn-79 mutant receptor demonstrates no in- crease in [3H]yohimbine dissociation rate upon the addition of Na’ (Fig. 2b). Interestingly, the [3H]yohimbine dissociation rate in the absence of Na+ resembles that for wild type receptors assayed in the presence of Na’. This more rapid rate of t3H]yohimbine dissociation in the Asn-79 mutant even in the absence of Na+ may result from a change in the receptor structure that fosters a conformation characteristic of the more rapidly dissociating form of the Na’-occupied wild type Lu2-receptor. Whatever the explanation for the intrinsic kinetic properties of the Asn-79 mutant, the inability of adrenergic binding to the Asn-79 mutant to be modulated by Na+ cannot result from simple charge manipulation of the hydrophobic, transmembrane domains of the receptor protein, since neither Asn-130 nor Asn-432 mutants display altered rates of disso- ciation upon the loss of a negatively charged residue.

Another characteristic effect of Na’ on cuz-receptor-ligand interactions is the ability of Na’ to induce an overall increase in receptor affinity for antagonists. Na+ facilitates both the dissociation rate of [3H]yohimbine from the az-adrenergic receptor, as well as the association rate. Since there is greater effect on the association rate, there is an overall increase in affinity of the Luz-receptor for the antagonist caused by Na’ (2). In addition, there is an increase apparent in density of a*-receptors when Na+ is present (2, 3, and data not shown for expressed porcine az-receptors). When [3H]yohimbine sat- uration binding is monitored, wild type az-receptors as well as Asn-130 and Asn-432 mutants (Fig. 2, e, g, and h) exhibit an approximately 2-3-fold increase in receptor affinity for the antagonist when Na’ is present in the incubation. In contrast, Na’ influences neither the affinity (Fig. 2fl nor the apparent density of t3H]yohimbine binding to the Asn-79 mutant recep- tors.

The effects of Na’ on a2-receptor-agonist interactions were evaluated by monitoring epinephrine competition for [3H] yohimbine binding. As shown in Fig. 3, the ability of Na’ to decrease receptor affinity for epinephrine characteristic of wild type receptors (Fig. 3~) is not observed for the Asn-79 mutant (Fig. 36), whereas Na’ modulation of receptor affinity for epinephrine is observed for both Asn-130 and Asn-432 receptor mutants (cJ legend to Fig. 3).

The findings in Figs. 2 and 3 indicate that all parameters for monitoring allosteric effects of Na+ on olz-receptor-ligand interactions, i.e. acceleration of the rate of [3H]yohimbine dissociation, increase in the affinity (decrease in Kn) for [3H] yohimbine binding, and an increase in the EC&,, for agonist competition for radiolabeled antagonist binding, are lost when Asp-79 is mutated to Asn-79 in the porcine at-receptor gene. These data demonstrate that the reciprocal effects of Na’ on agonist versus antagonist binding are mediated via a common allosteric site.

TO assess whether the mutation of Asp-79 to Asn-79 was pleiotropic for other allosteric modulators, we analyzed rates

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

21594 cup-Receptor Mutant Lacks Allosteric Effects of Na’

TABLE II Aspartate equivalent to Asp-79 of the a u-receptor is conserved among all G-protein-coupled receptors

Receptor Ref. Predicted transmembrane 2 sequence Effectors Human p-2 (40) NYFITSLACA D LVMGLAWPFGAAHILMK Human p- 1

t Adenylyl cyclase (41) NLFIMSLASA D LVMGLLWPFGATIWWG

Porcine a-2 A (19) NLFLVSLASA D ILVATLVIPFSLANEVMG Human n-2 A (36) NLFLVSLASA D ILVATLVIPFSLANEVMG Human (u-2 C 4 (37) NLFLVSLASA D ILVATLVMPFSLANELMA J Adenylyl cyclase Rat (u-2 B (36) NLFLVSLAAA D ILVATLIIPFSLANELLG t K+ channels Human dopamine-2 (42) NYLIVSLAVA D LLVATLVMPWWYLGWG 1 Ca’+ channels Human muscarinic-2 (43) NYFLFSLACA D LIIGVFSMNLYTLYTVIG Hamster a-1 (39) NYFIVNLAIA D LLLSFTVLPFSATLEVLG Human muscarinic-1 (43) NYFLLSLACA D LIIGTFSMNLYTTYLLMG t Phospholipase C Bovine substance K (44) NYFIVNLALA D LCMAAFNAAFNFWASHN Bovine opsin (30, 31) NYILLNLAVA D LFMVFGGFTTTLYTSLHG t cGMP phosphodiesterase

of [3H]yohimbine dissociation in the presence of amiloride analogs. Amiloride has been reported to affect binding to several G-protein-coupled receptors (26-28), but the allosteric uersus competitive nature of this influence has only been investigated in detail for az-adrenergic receptors (4, 5, 15-17, 19). Since allosteric modulation by amiloride analogs is re- tained in the “hydrophobic tryptic core” of the cu2-receptor (16) and, at least for Na+/H+ exchange proteins, there is a functional antagonism between Na’ and amiloride analogs (28, 29), it was important to determine whether amiloride regulation is retained in the mutant receptors. Ligand binding to all of the mutant receptors studied (Fig. 2, j, lz, and I) is modulated by amiloride analogs in a manner indistinguishable from the effects of these analogs in the wild type receptor. Thus, the rate of [3H]yohimbine dissociation from the mutant receptors is accelerated by 5-amino-substituted analogs of amiloride, e.g. ethylisopropylamiloride but not by guanidino- substituted analogs, e.g. chlorobenzyldimethylbenzamil. The finding that [3H]yohimbine binding to the Asn-79 mutant is modulated by amiloride analogs but not by Na’ indicates that the sites of interaction for these regulators occur in different domains of the cY,-adrenergic receptor. This conclusion is further supported by the observation that Na’ does not mask the effects of ethylisopropylamiloride on the rate of [3H] yohimbine dissociation (3, and data not shown for wild type cYz-receptor when expressed in COSMG cells).

The requirement for Asp-79 in Na+ regulation of az-adre- nergic receptors implies a crucial function for this allosteric regulation, as the predicted amino acid sequences of G-pro- tein-coupled receptors demonstrate the invariance of an as- partate at this topographical position in the second transmem- brane domain (ct Table II). In contrast, Asp-130, although invariant at this topographical position in all G-protein- coupled receptors for hormones and neurotransmitters, is changed to glutamate in bovine opsins (30, 31). Likewise, the existence of an aspartate residue at the same topographical position as predicted for Asp-432 in the a*-receptor occurs in many (19, 36-38, 40, 41, 43), but not all (30, 31, 44), G- protein-coupled receptors. The high degree of conservation of Asp-79 among G-protein-coupled receptors has made it a target for mutagenesis in previous reports. For example, sub- stitution of this conserved Asp with Asn in &adrenergic (18, 32) or M,-muscarinic (33) receptors resulted in little or no change in receptor affinity for antagonists but altered receptor affinity for agonists, the extent of which corresponded to the efficacy of the agonist (18, 32, 33). These findings resemble, phenomenologically, those for Na+ regulation of receptor- ligand interactions at cuz-receptors, where the extent of de-

crease in receptor affinity for agonists by Na’ parallels agonist efficacy in inhibiting adenylyl cyclase activity (1).

SUMMARY

We conclude that Asp-79 is directly involved in Na’ regu- lation of receptor-ligand interactions at cuz-adrenergic recep- tors with the likely mechanism being that Na’ binds to the carboxylate form of this residue. The invariance among the entire family of G-protein-coupled receptors of an aspartate residue at the topographical position of Asp-79 in the LYE- receptor implies that allosteric regulation of receptor-ligand interactions by Na’ may have a critical role in receptor function. The identification of a single point mutation, such as Asp-79 to Asn-79, that eliminates Na+ regulation while retaining ligand binding and allosteric modulation of that binding by amiloride analogs will be an important tool for investigators in a variety of fields. Future studies can address the role of Na+ in coupling of G-protein receptors to their multiple effecters via diverse GTP-binding proteins (34, 35) and, ultimately, the role of Na+ in mediating the myriad physiological effects evoked by these receptors.

Acknowledgments-We thank Drs. David L. Garbers and Graham Carpenter for reading the manuscript.

REFERENCES 1. Tsai, B. S., and Lefkowitz, R. J. (1978) Mol. Pharmacol. 14, 540-

548 2. Limbird, L. E., Speck, J. L., and Smith, S. K. (1982) Mol.

Pharmacol. 21, 609-617 3. Nunnari, J. M., Repaske, M. G., Brandon, S., Cragoe, E. J., Jr.,

and Limbird, L. E. (1987) J. Biol. Chem. 262, 12387-12392 4. Howard, M. J., Hughes, R. J., Motulsky, H. J., Mullen, M. D.,

and Insel, P. A. (1987) Mol. Phurmacol. 32,53-58 5. Jagadeesh, G., Cragoe, E. J., Jr., and Deth, R. C. (1990) J.

Pharmacol. Exp. Ther. 252,1184-1196 6. Neve, K. A., Henningsen, R. A., Kinzie, J. M., De Paulis, T.,

Schmidt, D. E., Kessler, R. M., and Janowsky, A. (1990) J. Pharmacol. Exp. Ther. 252,1108-1116

7. Pert, C. B., and Synder, S. H. (1974) Mol. Pharmacol. 10, 868- 879

8. Blume, A. J., Lichtshtein, D., and Boone, G. (1978) Proc. Natl. Acad. Sci. U. S. A. 76.5626-5630

9. Kosterlitz, H. W., Pateison, S. J., and Robson, L. B. (1988) J. Recept. Res. 8, 363-373

10. Nagao, M., Sakamoto, C., Matozaki, T., Nishizaki, H., Konda, Y., Nakano, O., and Baba, S. (1989) Nippon Naibumpi Gakkai Zasski 65. 1357-1366

11. Jakobs, K. g. (1979) Mol. Cell. Endocrinol. 16, 147-156 12. Jakobs. K. H.. Aktories. K.. and Schultz, G. (1981) Adu. Cyclic

Nu&otide ies. 14, 173-187 13. Minuth, M., and Jakobs, K. H. (1986) Naunyn-Schmeidebergs

Arch. Pharmakol. 333, 124-129 14. Limbird, L. E. (1984) Am. J. Physiol. 247, E59-E68

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

cr2-Receptor Mutant Lacks Allosteric Effects of Na+

15. Limbird, L. E., and Speck, J. L. (1983) J. Cyclic Nucleotide and 32. Chung, F., Wang, C., Potter, P. C., Venter, J. C., and Fraser, C. Protein Phosphorylation Res. 9, 191-201 M. (1988) J. Biol. Chem. 263,4052-4055

16. Wilson, A. L., Guyer, C. A., Cragoe, E. J., Jr., and Limbird, L. E. 33. Fraser, C. M., Wang, C., Robinson, D. A., Gocayne, J. D., and (1990) J. Biol. C&m. 265,17318-17322 Venter, J. C. (1989) Mol. Phnrmacol. 36,840-847

17. Motulsky, H. J., and Insel, P. A. (1983) J. Biol. Chem. 258, 34. North, L. A., and Surprenant, A. (1985) J. Physiol. 358, 17-33 3913-3919 35. Dunlap, K., Holz, G. G., and Rane, S. G. (1987) Trends Neurosci.

18. Strader, C. D., Sigal, I. S., Register, R. B., Candelore, M. R., 10,241-244 Rands, E., and Dixon, R. A. F. (1987) Proc. Natl. Acad. Sci. U. 36. Kobilka, B. K., Matsui, H., Kobilka, T. S., Yang-Feng, T. L., S. A. 84,4384-4388 Franke, U., Caron, M. G., Lefkowitz, R. J., and Regan, J. W.

19. Guyer, C. A., Horstman, D. A., Wilson, A. L. Clark, J. D., Cragoe, (1987)Science 238,650-656 E. J., Jr., and Limbird, L. E. (1990) J. Biol. Chem. 265,17307- 37. Regan, J. W., Kobilka, T. S., Yang-Feng, T. L., Caron, M. G., 17317 Lefkowitz, R. J., and Lobilka, B. K. (1988) Proc. Nutl. Acud.

20. Zoller, M. J., and Smith, M. (1983) Methods Enzymol. 100, 468- 500

21. Esser, V., Limbird, L. E., Brown, M. S., Goldstein, J. L., and Russell, D. W. (1988) J. Biol. Chem. 263, 13282-13290

22. Andersson, S., Davis, D. L., Dahlback, H., Jornvall, H., and Russell, D. W. (1989) J. Biol. Chem. 264,8222-8229

23. Gorman, C. (1986) in DNA Cloning Vol. ZZ, A Practical Approach (D. M. Glover, ed) pp. 143-164, IRL Press, Oxford

24. Lanier, S. M., Graham, R. M., Hess, H. J., Grodski, A., Repaske, M. G., Nunnari. J. M.. Limbird. L. E.. and Homcv. C. J. (1986) Proc. iutl. Acud. Sci. ‘U. S. A. 83, 9358-9362 -’

25. Howard, M. J., Mullen, M. D., and Insel, P. A. (1981) Am. J. Physiol. 253, F21-F25

26. Haussinger, D., Brodde, O., and Starke, K. (1987) Biochem. Pharmacol. 36,3509-3515

27. Martinez, J. R., Barker, S., and Camden, J. (1989) Eur. J. Pharmacol. 164,335-339

28. Kinsella, J. L., and Aronson, P. S. (1981) Am. J. Physiol. 241, F374-F379

29. Mahnensmith, R. L., and Aronson, P. S. (1985) J. Biol. Chem. 260,12586-12592

30. Nathans, J., and Hogness, D. S. (1983) Cell 34, 807-814 31. Pappin, D. J. C., and Findlay, J. B. C. (1984) Biochem. J. 217,

605-613

Sci. U. S. A. 85,6301-6305 38. Zeng, D., Harrison, J. K., D’Angelo, D. D., Barber, C. M., Tucker,

A. L., Lu, Z., and Lynch, K. R. (1980) Proc. N&l. Acud. Sci. U. S. A. 87,3102-3106

39. Cotecchia, S., Schwinn, D. A., Randall, R. R., Lefkowitz, R. J., Caron, M. G., and Kobilka, B. K. (1988) Proc. Natl. Acud. Sci. U. S. A. 85, 7159-7163

40. Kobilka, B. K., Frielle, T., Dohlman, H. G., Bolanowski, M. A., Dixon, R. A. F., Keller, P., Caron, M. G., and Letkowitz, R. J. (1987) J. Biol. Chem. 262, 7321-7327

41. Frielle, T., Collins, S., Daniel, K., Caron, M. G., Lefkowitz, R. J., and Kobilka, B. K. (1987) Proc. Nutl. Acad. Sci. U. S. A. 84, 7920-7924

42. Grandy, D. K., Marchionni, M. A., Maham, H., Stofko, R. E., Alfano, M., Frothingham, L., Fischer, J. B., Burk-Howie, K. J., Bunzow, J. R., and Server, A. C. (1989) Proc. Nutl. Acud. Sci. U. S. A. 86,9+62-9766

43. Peralta. E. G.. Ashkenazi. A.. Winslow. J. W.. Simon. D. H.. Ramachandran, J., and Capon, D. J. (1987) EMBO J. 6,3923- 3929

44. Masu, Y., Nakayama, K., Tamaki, H., Harada, Y., Kuno, M., and Nakanishi, S. (1987) Nature 329, 836-838

45. Bylund, D. B., Ray-Prenger, C., and Murphy, T. J. (19811) J. Phurmucol. Exp. Ther. 245,600-607

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from

D A Horstman, S Brandon, A L Wilson, C A Guyer, E J Cragoe, Jr and L E Limbirdalpha 2-adrenergic receptors by sodium.

An aspartate conserved among G-protein receptors confers allosteric regulation of

1990, 265:21590-21595.J. Biol. Chem. 

  http://www.jbc.org/content/265/35/21590Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

to choose from all of JBC's e-mail alertsClick here

  http://www.jbc.org/content/265/35/21590.full.html#ref-list-1

This article cites 0 references, 0 of which can be accessed free at

by guest on September 28, 2020

http://ww

w.jbc.org/

Dow

nloaded from