Receptors for pituitary adenylate cyclase-activating polypeptide Comparison with vasoactive intestinal peptide receptors

Receptors for Pituitary Adenylate Cyclase-Activating Polypeptide Comparison with Vasoactive Intestinal Peptide Receptors Akira Arimura

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a new member of the secretin-glucagon-vasoactive intestinal peptide (VIP) family ofpeptides, being most homologous to VIP. PACAP exists in two amidated forms with 38 residues (PACAP38) and 27 residues (PACAP27), respectively. PACAP is the major form in tissues. There are two types of high-affinity receptors for PACAP: type I, which specifically binds to both PACAPs, and type II, which is shared with VIP. vpe I PACAP receptors appear to have two subtypes: type IA, which binds to both PACAP and PACAP27, with slight preference for the latter, and type IB, with greater preference for PACAP38. Distribution of the type I PACAP receptor is different from that of VIP, and it is found in high concentrations in brain, spinal cord, anterior pituitary, adrenal medulla, spermatogonia at certain stages, mature spermatozoa, and some cell lines. Type II PACAP receptors are found in lung, liver, intestine, and other tissues, and their distribution is similar to that of the VIP receptor. Type II PACAP receptor might be similar to or identical with the VIP receptor. (Trends Endocrinol Metab 1992;3:288-294)

BRIEF REVIEWS

tion, but also the function of various other tissues and organs.

Like other peptide hormones, PACAP exerts its biologic activity through a specific receptor on its target cells. Fur- thermore, in view of the considerable homology in the primary structure and certain similar biologic activity, my lab- oratory and others have studied the PACAP receptor in comparison with the VIP receptor. The current status of the biologic actions of PACAP has been recently reviewed (Arimura 1992). This review discusses recent findings with regard to the identification, distribution, specificity, and affinity of the PACAP receptor and its subtypes.

?? Characterization, Distribution, and Signal Transduction of PACAP-Binding Sites

Characterization and Distribution

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a newly recog- nized member of the secretin-glucagon- vasoactive intestinal peptide (VIP) family of peptides (Table 1). It was isolated fi-om ovine hypothalamic tissues during an attempt to discover a novel hypophysi- otropic factor, using adenylate cyclase activation in the cultured rat pituitary cells as the biologic response index (Miyata et al. 1989). PACAP is present in two amidated forms, one with 38 residues (PACAP38) and the other with 27

residues (PACAP27). The N-terminal 28 amino acids of PACAP show a 68% sequence homology with VIP. Although certain bioactivity, including the va- sodepressor activity, of PACAP is similar to that of VIP, there are considerable qualitative and quantitative differences between PACAP and VIP in other biologic activities.

Akira Arimura is at the Department of Medi- cine, Tulane University School of Medicine, New Orleans, LA 70012; and the US-Japan Biomedical Research Laboratories, Tulane University Hebert Center, Belle Chasse, LA 70037, USA.

The magnitudes of adenylate cyclase- stimulating activity of PACAP and PACAP are similar, but are nearly lOOO-10,000 times greater than that of VIP, as determined in rat pituitary cell cultures (Miyata et al. 1989). PACAP also increases CAMP accumulation in cultured rat neurons, astrocytes (Arimura et al. 1990), a pancreatic cell line (Robbe- recht et al. 1991a), and other tissues and cell lines. These findings suggest that PACAP regulates not only pituitary func-

Following isolation of the peptides, ef- forts were made to characterize the binding sites for PACAP and describe their distribution in various tissues. In freshly prepared rat anterior pituitary membranes, [12sI]PACAP27 binding was specific and saturable and the data were consistent with a single high-affinity binding site (K,=O.S-2.0 nM). Unlabeled PACAP and PACAP completely dis- placed the binding, with similar affinity, suggesting that PACAP competes for an identical population of [’ 251]PACAP27- binding sites (Gottschall et al. 1990; Lam et al. 1990). VIP and related and unrelated peptides did not affect binding (Figure 1). PACAP and VIP compete for a binding site in some tissues, whereas PACAP-specific binding sites are present in other tissues. An almost identical relative magnitude of binding was observed between PACAP and VIP in lung, liver, duodenum, ovary, and thymus (Figure 2). However, whereas PACAP binding to hypothalamic and anterior pituitary membranes was great, VIP binding to these tissues was negligi- ble. VIP, at near micromolar concentrations, was unable to displace [ * 251]PACAP27 binding in hypothalamus and pituitary, yet VIP was highly potent

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Table 1. The amino acid sequences of PACAP and its related peptides

Species Pepiide Amino acid sequence

I 5 10 15 20 25 30 35 40 45

PACAP R-S-D-G-I-F-T-D-S-Y-S-R-Y-R-K-Q-M-A-V-K-K-Y-L-A-A-V-~G-K-R-Y-K-Q-R-V-K-N-K-~

PACAP S-S-D-G-I-F-T-D-S-Y-S-R-Y-R-K-Q-M-A-V-K-K-Y-L-A-A-V-L-~ VIP E-S-D-~-~-F-T-D-~-Y-r-R_L-R-R-q-M-A-v-K-Q-M-A-V-K-K-Y-L-~-~-~-L-~-~ PHI R-a-D-G-V-F-T-~-~-Y-S-R-~-~-~-Q-L-S-A-K-K-Y-L-E-S-L-~-~ Helodermin S-S-D-B-I-F-T-Q-O-Y-S-K-L-L-A-K-L-A-L-Q-K-Y-L-A-S-I-L-G-~-R-T-S-~-F-~-* _- ------ -- -- ___--- Secretin E-S-D-G-~-F-T-~-~-L-S-R-L-R_E-G-A-R-L-Q-R-L-L-Q-G-L-~-~ -------- Glucagon E-S-Q-G-T-F-T-S-D-Y-S-g-Y-L-n-S-R-R-A-O-Q-F-Y-~-U-L-M-N-T ------------- GHRH-(1-44) Y-B-D-a-I-F-T-N-s-Y-~-~-~-~-~-Q-L-5-B_BE-A-K-L-* _-_--- GIP ~-~-D-G-~-F-I-S-D-Y-S-~-~-~-~--~-~-Q-O_LI-E-Y-hl_bi-L-L-B-q-g ---

Differences from PACAP are underlined. o, ovine; b, bovine; g, Gila monster: p, porcine; h, human; *, NH,; PHI, peptide histidine isoleucine; and GE’, gastric lnhibitoq polypeptide

(IC 50=3 nM (although -3-10 times less potent than PACAP or PACAP38) in displacing [ 1 2sI]PACAP27 binding in lung membranes (Figure 3; Gottschall et al. 1990). In cultured splenocytes, it was confirmed that PACAP and VIP share a binding site, since identical binding af- finities and numbers of sites were obtained with either [*251]PACAP27 or [t251]VIP as the labeled ligand (Tatsuno et al. 1991a; Ottaway and Greenberg 1984). These data support the existence of a distinct PACAP-specific binding site and the existence of a binding site that PACAP may share with VIP. After subse- quent investigation (Shivers et al. 1991; Robberecht et al. 1991a), these sites were named the type I (PACAP-specific site) and type II (PACAP and VIP shared site) PACAP receptors. The expression of these

sites is not tissue specific, and the pro- portion of each site in a variety of tissues has been characterized.

Several receptor autoradiography and radioligand-binding studies have described the tissue distribution for each type of PACAP receptor. Using receptor autoradiography in the brain, dense populations of type I sites were localized to the hypothalamus, hippocampus, sub- stantia nigra, septum, and cerebellum (Masuo et al. 1992). [t*sI]PACAP27 binding has been demonstrated by receptor autoradiography in testis, epididymis, adrenal medulla, lung, liver, prostate gland, and seminal vesicle, but specific binding was not detected in heart, kidney, or thymus (Shivers et al. 1991). Each of these tissues appears to express varying proportions of type I and type II

Figure 1. Representative displacement curves of [1251]PACAP27 binding by unlabeled PACAP27, PACAP38, and PACAP380H in rat anterior pituitary membranes. The figure also demonstrates the specificity of [ 1251]PACAP27 binding with m-related peptides and unrelated peptides. Anterior pituitary membranes (41pg) were incubated with 124 pM of [1251]PACAP27 with increasing concentrations of unlabeled hormones at 22°C for 90 min. Results are expressed as a percentage of binding in the absence of unlabeled hormone. PHI, peptide histidine isoleucine; IL-l, interleukin-1; and EGF, epidermal growth factor. From Gottschall et al. (1990).

g E loo- . x.

* M+ ?? PACAP

0 PACAP35OH o PACAP

+ PACAPCysZ3(24-38) A VIP

h Secle,i”

. glucagon

* PHI

. LHRH

?? IL-1

* EGF

01 .l 1 IO 100 1000

DOSE OF PEPTIDE (nM)

PACAP-binding sites. For example, tissues that contain predominantly type I receptors (60% to nearly 100%) include the adrenal gland, testicular germ cells, anterior pituitary, hypothalamus and other brain regions, spinal cord, and cultured astrocytes. Tissues that contain predominantly type II receptors (80% to nearly 100%) include lung, liver, prostate gland, seminal vesicle, and cultured splenocytes (Gottschall et al. 1990; Rob- berecht et al. 1991a; Gottschall et al. 1991; Shivers et al. 1991; Tatsuno et al. 1991a). A binding site for PACAP that has a 1000 times lower affinity than the high-affinity site exists in cultured rat astrocytes and splenocytes (Tatsuno et al. 1990 and 1991a). Classic membrane radioligand-binding assays have failed to detect [tz51]PACAP27 binding to whole pancreas or testis (our unpublished ob- servations; Lam et al. [ 19901). However, a dense, localized population of binding sites has been identified in testis with tissue receptor autoradiography (Shivers et al. 1991), and PACAP-binding sites were demonstrated in a pancreatic acinar cell line (Buscail et al. 1990). Thus, the use of whole tissues for classic membrane radioligand assays may mask specific binding that occurs in localized areas of the tissue. In summary, several tissues, including brain and anterior pituitary membranes, contain high levels of the type I PACAP-binding site, whereas lung and liver are prototype tissues that contain high levels of the type II PACAP-binding site.

Using [1251]PACAP38 as the labeled ligand, Cauvin et al. (1991) described a population of binding sites in several

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AP HYPO ADR KID LIV LUN DVO Irm OK=, THY

Figure 2. Distribution of [1251]PACAP27 and [1251]VIP in various tissue membranes. About 120 pM of [*25I]PACAP27 (n=4) or [12sI]VIF (n=3) was incubated with different tissue membranes in the absence or presence of 200 nM of the appropriate unlabeled peptide, PACAF27 or VIP, at 22% for 90 min. Results are expressed as the mean + SEM, calculated by the difference between frnol bound in the absence of unlabeled peptide and in the presence of unlabeled peptide. The mean protein levels for seven assays are anterior pituitary (AP), 19 pg; hypothalamus (HYF’O), 28 pg; adrenal (ADR), 27 pgg; kidney (IUD), 25 pg; liver (LIV), 49 pg; lung (LUN), 15 pg; duodenum (DUO), 40 pg; uterus (UTR), 9 pg; ovary (OVR), 30 pg; and thymus (THY), 49 pg. From Gottschall et al. ( 1990).

brain regions that preferentially binds PACAP compared with PACAP27. This site was named the type B PACAP receptor to distinguish it from a site in brain tissues that binds both PACAP and PACAP with similar affinity and was named the type A PACAP receptor. Since both of these sites do not significantly bind W, they may better be termed types IA and IB. In addition, these two populations of PACAP-specific binding sites were shown to exist on the pancreatic acinar cell line AI-~-2J (Robberecht et al. 1991 b). To our knowledge, other

Figure 3. Representative displacement curves of [1251]PACAP27 binding by unlabeled PACAP27, PACAP38, and VIP in lung membranes. Lung membranes (17 lg) were incubated with 124 pM of [1251]PACA127 with increasing concentrations of unlabeled hormones at 22°C for 90 min. Results are expressed as percentage of binding in the absence of unlabeled hormone. From Gottschall et al. (1990).

o PACAP(l-38)NH2

PACAP(l -27)NH2

.Ol .l 1 10 100 1000

DOSE OF PEPTIDE (nM)

tissues have not been screened for A and B subtype distribution.

Type I PACAP-binding sites also exist on two neuronal-like cell lines: the neuroblastoma cell line NB-OK (Cauvin et al. 1990) and the pheochromocytoma cell line PC12 (Watanabe et al. 1990). W and other peptides of the secretin- glucagon family either had a weak affinity for these binding sites (300-1000 times less potent than PACAP27) or did not bind to these sites.

Signal Transduction, Biologic Activity, and PACAP Analogues

PACAP receptors have been shown to be linked to adenylate cyclase in several cell types and cell lines. Both PACAP peptides stimulate adenylate cyclase in cultured anterior pituitary cells, astrocytes, and neurons (Arimura et al. 1990). There is some controversy as to the cellular localization of binding sites on cultured pituitary cells; however, it seems clear that PACAP- binding sites are present on all cell types, including folliculostellate cells, but the population of PACAP receptor-containing cells in each cell type varies (Vigh et al. 1992). In addition, PACAP raises intracellular Ca*+ in cultured pituitary cells (Vigh et al. 1992; Canny et al. 1992).

Interestingly, it was recently demonstrated that, although PACAP and PACAP exhibit similar potency in ele- vating CAMP levels in PC12 cells, PACAP was lOO- to IOOO-fold more potent than PACAP in stimulating inositol phospholipid turnover in the same cell line (Deutsch and Sun 1992). Therefore, it is possible that in PC12 cells the inositol phosphate cascade is stimulated through PACAP38-preferring receptors (type IB), whereas type IA receptors could be linked with adenylate cyclase. In a cell line that contains mainly high-affinity type II PACAP receptors, the GH, pituitary cell line, PACAP27, PACAP38, and W exhibited similar potency in increasing CAMP levels. Concentrations of PACAP of up to 1 PM, however, failed to stimulate inosi- to1 phospholipid turnover in GH, cells, suggesting that type II PACAP receptors or W receptors are not linked with the inositol phosphate cascade (Deutsch and Sun 1992).

In liver membranes, which contain mainly type-II-binding sites, both PACAP and PACAP stimulated adenylate cyclase with the same low efficacy as W (Waelbroeck et al. 1981), causing only a twofold increase. The concentration required for half-maximal stimulation with both PACAP peptides was identical (0.02 nM), but slightly lower than that for W (0.05 nM) (Robberecht et al. 1991a). In addition, mainly type II PACAP receptors are present in cultured splenocytes. Both PACAP peptides and W exhibited similar potency in inhibit- ing concanavalin-A-stimulated splenocyte proliferation (Tatsuno et al. 1990).

Type I PACAP receptors on adrenal Gourlet et al. (199 la) showed competi- chromaffin cells are activated by PACAP tion binding curves by using ([t*sI]acetyl- or PACAP38, resulting in a rapid release Hisl)PACAP27 as a radioligand and dose- of epinephrine: 10 nM of PACAP or effect curves of adenylate cyclase PACAP was as efficacious in stimulat- activation in human SUP-T1 lympho- ing epinephrine secretion as 100 J.~M of blastic membranes. Both PACAP and

acetylcholine (Watanabe et al. 1992), and the stimulation by PACAP was more sustained compared with acetylcholine. In addition, PACAP and PACAP raised intracellular CAMP and Ca*+ concentrations. Like chromaffin cells, PC12 cells bind and are activated by PACAP (Watanabe et al. 1990). Mainly type I PACAP-specific receptors are present on PC12 cells. Both PACAP and PACAP increase intra- and extracellular CAMP to a similar extent, and W is -1000 times less potent (Watanabe et al. 1990).

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PACAP stimulated adenylate cyclase through a single class of helodermin- preferring VIP receptors with the following order of potency: helodermin=(acetyl- Hisi)PACAP27 >PACAP38 >PACAP27 > VIP. PACAP(6-27) and (des His’, Asn3)PACAP27, at concentrations from 0.5 to 2.0 l.tM, acted as competitive antagonists (and weak agonists). Using a series of 13 PACAP analogues and fragments and three VIP analogues, these authors identified positions 1, 2, 3, 9, and 13 in PACAP as being important for high-affinity binding (Gourlet et al. 1991b).

?? Molecular Analysis of PACAP and VIP Receptors

Cross-linking procedures were used to analyze the molecular characteristics of PACAP receptors in various tissues and cell lines. Major binding proteins pos- sessed molecular weights ranging from 57,000 to 65,000, depending on tissues and cell lines. Since type II PACAP receptors present in lung, liver, and other peripheral tissues may be similar or identical with VIP receptors, it is appropriate to deal first with type I PACAP receptors when the molecular analysis of specific PACAP receptors is attempted. As discussed earlier, tissues and cell lines that possess type-I-binding sites as the major receptor subtype include brain, pituitary, spinal cord, adrenal medulla, testicular germ cells, spermatozoa, rat pancreatic acinar cell line AR4-2R, human neuroblastoma cell line NB-OK, and rat pheochromocytoma cell line PC12.

Gottschall et al. (1991) cross-linked [tz51]PACAP27 to rat hypothalamic membranes by using the amine-reactive, water- soluble, homobifunctional cross-linker BS3. The cross-linked complexes were solubilized in SDS buffer and subjected to electrophoresis on SDS-PAGE by using 10% gels. A major band of 60 kD was evident, but a minor band near the origin of the gel, a heavy component, was also present; and 500 nM of unlabeled PACAP or PACAP completely eliminated both the major 60-M) band and the minor band near the gel origin, whereas preincubation with VIP or PACAP(l-23) did not (Gottschall et al. 1991). The minor band, a much larger molecule than 60 kD, might represent the PACAP receptor associated with a G

protein. Whether both components have a similar sensitivity to GTP was not investigated. Furthermore, these authors showed that binding of [1251]PACAP27 to rat hypothalamic membranes was par- tially inhibited by GTP and nonhy- drolyzable GTP analogues, suggesting that the PACAP receptors are associated with G protein. Assuming that one molecule of PACAP binds to one molecule of the protein, the PACAP receptor itself has an apparent molecular mass of 57 kD. Tatsuno et al. (1991b), using the same cross-linker and [1251]PACAP27 as the radioligand, demonstrated a PACAP- binding protein with a molecular mass of 57 kD in rat astrocyte membrane preparations. Ohtaki et al. (1990) used three different cross-linkers, disuccin- imidyl suberate (DSS), N-hydroxysuc- cinimidyl-4-azidobenzoate (HSAB), and N-succinimidyl-6(4’-azido-2’-nitrophen- ylamino)hexanoate (SANPAH), for affinity labeling of the PACAP receptor in the bovine brain with [1251]PACAP27. The molecular mass of the major band on SDS-PAGE was again estimated to be 60 kD, regardless of the identity of the cross-linker. The labeling density of the 60-kD band decreased with increasing concentrations of unlabeled PACAP, whereas it did not decrease in the presence of VIP. The labeling density of the 60-kD band decreased in the presence of GTP, but not in the presence of ATP. Buscail et al. (1990) using [1251]PACAP27 as the radioligand and DSS as the cross- linker, demonstrated on SDS-PAGE electrophoresis a major band of receptor- ligand complex with a molecular mass of 68 kD in the AR 4-2J cell line. Therefore, the receptor itself may have a molecular mass of 65 kD. Tracer bound to the AR 4-2J ceil membranes after 20 min disso- ciated with a t,,* of 20 min, as studied by isotopic dilution. This tl,l was transiently reduced to 4 min in the added presence of 10 uM GTP. The PACAP receptors in human neuroblastoma cell line NB-OK membranes were also cross-linked with [1251]PACAP27 and DSS as the cross- linker, and the molecular mass of the receptor-ligand complex was estimated by SDS-PAGE and autoradiography (Cau- vin et al. 1990). The major band corre- sponded to a 68-kD protein. Thus, the receptor itself should have a molecular mass of 65 kD.

Masuda et al. (1990) reported the solubilization of unoccupied PACAP re-

ceptors from bovine brain membrane preparations. Seven different detergents were used for solubilizing the PACAP receptor. These authors found that CHAPS (0.5%) was superior to other detergents for solubilizing functional PACAP receptors. Binding saturation experiments using [12sI]PACAP27 revealed that the solubilized extract contained a single class of binding sites, with a Kd of 200 pM and a B,,, of 0.9 pmol/mg protein. The q did not change significantly after solubilization. Competitive binding experiments confirmed that the solubilized receptor retained its specificity for PACAP. The molecular size and the sensitivity to GTP of the solubilized protein have not been investigated.

On the other hand, the molecular mass of major VIP receptors ranged from 46 to 73 kD: 46- or 47-kD species were obtained from rat brain membranes (Couvineau et al. 1986a; Vasiloff et al. 1986), human lymphoblastoma cell line Molt-46T (Wood and O’Dorisio 1985) and the pituitary tumor cell line GH, (Wood et al. 1985). Larger receptor species reported include 62 kD from human lung membranes, 53 kD from rat, 61 kD from rabbit, and 63 kD from guinea pig lung membranes (Dick- inson et al. 1986). Larger VIP receptor proteins were also demonstrated in rat intestinal epithelial membranes (73 kD; Laburthe et al. 1984) and human colonic epithelial membranes (63 kD; Couvineau and Laburthe 1985a). Although the molecular weights of PACAP and VIP receptors reported are in the same range, the PACAP receptors in rat brain membranes appear to be significantly larger than the VIP receptors in the same tissue.

Liver contains a high concentration of VIP receptors, -1 pmol/mg protein or more (Robberecht et al. 1984 and 1986), and liver membrane VIP-receptor complexes were used for solubilization and purification. Triton-X- 1 OO-solubilized liver [ 1251]VIP-binding protein complexes revealed two components: a major one (80%) and a minor one (20%). Both components are specifically labeled, since they almost completely disappeared when 0.3 uM of unlabeled VIP was added to the [1251]VIP. The molecular masses of these two components were 150 kD (heavy) and 52 kD (light). GTP induced a rapid dissociation of [**sI]vTp from the heavy component, whereas it did not affect the stability of the light one. Similarly, when GTP was added to each component in

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sucrose gradients, the heavy component (s 2,,,=6.0 s) was no longer observed after ultracentrifugation, whereas the light one (s~~,~=~.O s) was unaffected. The results suggest that the heavy species consists of the VIP receptor of 52 kD associated with a G protein, probably G,. This was further supported by the ability of the 150~kD species to generate the 52-kD component after mild urea treat- ment (Couvineau et al. 1986b). It was very similar to the putative VIP-binding protein of 51 kD (including VIP) identified by SDS-PAGE analysis after cross- linking of [12sI]VIP to liver membranes (Couvineau and Laburthe 1985b). Solubilization of unoccupied VIP receptors was also reported (Guerrero et al. 1986; Paul and Said 1987). [12sI]VIP was shown to bind solubilized material obtained from Lubrol-treated rat liver membranes (Guerrero et al. 1986). The molecular mass of the binding protein was estimated as 80 kD by chromatography on Sepharose 6B. Unfortunately, molecular sieving alone often yields a very inaccurate estimate of molecular mass for hydrophobic membrane proteins. Sensitivity to GTP of the solubilized protein was not examined. Paul and Said (1987) reported that VIP receptors from guinea pig lung were solubilized by CHAPS. Two binding components were identified, with Stokes radii of 5.9 nm and 2.3 nm, respectively. The major 5.9-nm component had a high affinity for VIP, and in the presence of high detergent concentration generated the 2.3-nm component, which had a low affinity for VIP. GTP inhibited the binding of [12sI]VIP to the 5.9-nm component, but not the 2.3-nm component. Thus, the major component solubilized from guinea pig lung by CHAPS com- pares well, with respect to Stokes radius and sensitivity to GTP, to the ternary complex of 150 kD (Stokes radius = 5.8 nm) solubilized from rat liver by Triton X-100 (Couvineau et al. 1986b).

Couvineau et al. (1990) solubilized pig liver VIP receptors with 6 mM CHAPS and purified this protein to homogeneity by one-step affinity chromatography by using a VIP-polyacrylamide resin. The purified receptor bound [i25I]VIP with a Kd of 22.3 nM, which was higher than that in liver membranes (0.17 nM), although it retained its peptide specificity toward VIP-related peptides. Uncou- pling of VIP receptors and G protein

during solubilization, at least in part, might account for the decrease of receptor affinity. This uncoupling was supported by the fact that VIP binding to receptors in solubilized extract was in- sensitive to GTP, whereas VIP binding to membrane-bound VIP receptors in liver and other tissues was sensitive to GTP (Laburthe and Couvineau 1988). How- ever, the reason why G protein remains associated with solubilized VIP receptors in some cases and not in others is unclear. This also appears to be the case for solubilized PACAP receptors. SDS- PAGE analysis of pig liver VIP receptor that was purified by VIP-polyacrylamide affinity chromatography revealed a single band with a molecular mass of 53 kD after either silver staining or radioiodi- nation. Affinity labeling of the purified receptor-[‘2sI]VIP complex with the use of DSP gave a single radioactive band that migrated with a molecular mass of 55 kD on SDS-PAGE. When the purified receptor was incubated with [ 12sI]VlP in the presence of an excess of unlabeled VI?, no radioactive band was observed (Couvineau et al. 1990).

?? Future Research of PACAP Receptors

The studies discussed above indicate the presence of high-affinity specific receptors for PACAP (type I) that do not bind VIP in the pituitary, brain, testis, adrenal medulla, and some tumor cell lines. Studies also suggest the presence of subclasses of type I PACAP receptors: types IA and IB. Type II PACAP receptors, which are similar or identical to VIP receptors, exist in various tissues, such as lung and liver. PACAP does not only activate adenylate cyclase and increase intracellular CAMP levels in various tissues, but it also stimulates the phosphatidyl inositol cascade and mobi- lizes intracellular Ca2+ (Canny et al. 1992; Deutsch and Sun 1992; Tatsuno et al. 1992; Watanabe et al. 1992). Although these activities are receptor-mediated effects, whether each of these effects is linked with a distinct, specific subclass of PACAP receptors remains unknown. Mobilization of intracellular Ca2+ by PACAP in cultured rat hippocampal neurons is not linked with an increase in CAMP levels or protein kinase A activation (Tatsuno et al. 1992). Accumulating data support the view that the PACAP

receptor is associated with a G protein. It is necessary to identify the G protein that triggers the activation of each of these effecters. The potency difference between PACAP and PACAP in the stimulation of phosphatidyl inositol in PC1 2 cells, despite the similar potency of these two forms of PACAP in CAMP accumulation, suggests distinct path- ways of signal transduction. Whether subclasses of PACAP receptors, that is, types IA and IB, are linked to distinct effecters remains to be investigated.

The abundance of type I PACAP receptors in the brain and solubilization of functional PACAP receptors have paved the way for possible purification and partial sequencing of the PACAP receptors. One-step affinity chromatography using a VIP-polyacrylamide resin re- sulted in 50,000-fold purification, yield- ing nearly pure VIP receptors from pig liver membranes (Couvineau et al. 1990). A similar method could also be applied for effective purification of PACAP receptors. It may not be too difficult to accumulate a few hundred picomoles of receptor protein to undertake microse- quencing of receptor fragments and subse- quent cloning of the receptor cDNA.

Cloning of PACAP receptor cDNA could also be conducted in a manner similar to that described for the cloning of a putative VIP receptor from Nalm 6 human leukemic pre-B cells and HT-29 human colon adenocarcinoma cells, both of which possess receptors specific for VIP (Sreedharan et al. 1991). A cDNA clone (GPRNl) encoding the human VIP receptor was identified in libraries prepared from the Nalm 6 line and the HT-29 cell line. The deduced 362-amino- acid polypeptide sequence encoded by GPRNl shares a seven-transmembrane segment hydrophobic@ profile with other G-protein-coupled receptors. COS-6 cells transfected with GPRNl bound [*251]VIP specifically with a Kd of 2.5 nM. It should be interesting to examine whether GPRNl- transfected COS-6 cells also bind [1251]PACAP with high affinity, and whether PACAP increases CAMP in these transfected cells. VIP stimulated adenylate cyclase in stably transfected Chinese hamster ovary Kl cells, inducing a three- fold increase in the intracellular level of CAMP. The VIP receptors cloned exhibit ~25% homology with other receptors in the same superfamily, and thus represent the subset of G-protein-coupled


receptors for VIP. However, Nagata et al. (1992) failed to confirm these findings, so they demand verification.

As this article was being prepared, a functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with secretin receptor cDNA (Isihara et al. 1992). This rat VIP receptor cDNA codes for 429 amino acids (without the signal peptide), has a calculated molecular weight of 48,946, and contains seven transmembrane segments. Unlike the above- described human cDNA, this rat cDNA is markedly homologous to secretin receptor cDNA. In addition, it shows signifi- cant sequence homology to the calcitonin and PTH receptors, suggesting that receptors for these four peptides constitute a new subfamily of G-protein-coupled receptors. When the cDNA was expressed in mouse COP cells, the transfected cells bound labeled VIP and the addition of VIP stimulated adenylate cyclase. PACAP competed for VIP binding and stimulated CAMP at concentrations similar to VIP. High levels of mPNA for the rat VIP receptor were detected in lung, liver, intestines, and brain (Isihara et al. 1992). Thus, this protein is certainly one subtype of several possible VIP receptors, maybe the type II PACAP receptor. Clearly, it is likely that soon the secretin and/or VIP receptor cDNAs may be used to clone a cDNA for the PACAP-specific (type I) receptor. The cloning, sequencing, and functional expression in host cells of PACAP receptors and their subclasses will lead to a focus on the structural determinants of these receptors for binding to respective ligands and for signal transduction.

?? Acknowledgments

The author thanks Drs. Paul Gottschall and Ichiro Tatsuno for critical reading and valuable suggestions for preparing this article. The findings at our laboratories were obtained during studies that were supported by National Institutes of Health grant DK09094 and a research grant from Takeda Chemical Industries.

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Receptors for pituitary adenylate cyclase-activating polypeptide Comparison with vasoactive intestinal peptide receptors