The cholecystokinin-gastrin peptide family appear to have evolved as ... octapeptide cholecystokinin 27-33 (CCKs) is the major CCK-neuropeptide with an.
Cellular and Molecular Neurobiotogy, Vol. I5, No. 5, 1995
Cholecystokinin Receptors Phil Boden, la M a t t h e w D . Hail, l and John H u g h e s ~ Received January 20, 1995; accepted February 23, 1995
INTRODUCTION The cholecystokinin-gastrin peptide family appear to have evolved as messengers intimately concerned with the intake and metabolism of nutrients (gastric motility and acid secretion, pancreatic exocrine and biliary secretion, and small bowel motility). During this evolution there was a parallel involvement with the nervous control of food ingestion and gastrointestinal transport and in common with other brain-gut peptide families shorters peptide fragments replaced the larger hormone species in the family as the predominant neural messengers. The octapeptide cholecystokinin27-33 (CCKs) is the major CCK-neuropeptide with an extensive distribution in the cerebral cortex, hypothalamus, hippocampus, olfactory lobes, basal ganglia, hind brain, the dorsal horn of the spinal cord and peripheral ganglia, and peripheral nerves. Although many of the central actions of CCK8 may be related to the control of metabolic balance studies with selective CCK antagonists also indicate that this neuropeptide and its receptors are also involved in many other diverse functions including learning, memory, stress responses, pain modulation, and possibly mood (Woodruff and Hughes, 1991). The development of selective CCK antagonists not only allowed a greater definition of the possible physiological roles of CCK but enabled the clear identification of two major CCK-receptor subtypes. The CCKA receptor was distinguished by its sensitivity to the sulphated form of CCK8 and the antagonist L364718 while the CCKB receptor is indifferent to the sulphation status of CCK and has high affinity for the antagonists L365260 and CI-988. Although it was originally thought that the receptor subtypes could also be distinguished on the basis of their anatomical distribution it is now clear that there is a considerable overlap in their distributions. The CCKA receptor although difficult to detect in rodent brain using binding studies is nevertheless 1 Parke-Davis Neuroscience Research Centre, Cambridge University Forvie Site, Robinson Way, Cambridge CB2 2QB, U.K. 2 To whom correspondence should be addressed. 545 0272.43,10/95/1001).0545507.50/0~) 1995PlenumPublishingCorporation
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readily demonstrable by electrophysiological techniques. Conversely the CCKB receptor is predominantly detected by binding studies in rodents but in primates there is a substantial component of CCKA receptor binding.
SIGNAL TRANSDUCTION MECHANISMS LINKED TO ACTIVATION OF CCK RECEPTORS
CCKB Receptors CCKB receptors are prevalent in the mammalian central nervous system and also in peripheral tissues including gallbladder, parietal and chief cells of gastric mucosa and gastrointestinal smooth muscle. At these peripheral sites they are often co-expressed with CCKA receptors making the interpretation of any mechanistic studies difficult unless selective agonists and antagonists are included. This, coupled with the increasing interest in the role of brain CCKB receptors, has meant that the majority of new information on the transduction processes linked to CCKB receptor activation has come from studies of CCK-sensitive neurones in brain slice preparations. The first evidence for a direct action of CCK on neurones was reported nearly 15 years ago by Dodd and Kelly (1981) who showed that CCK depolarized CA1 neurones in rat hippocampal slices by an apparent decrease in input resistance with a net equilibrium potential closer to zero than the cell resting membrane potential and suggestive of the involvement of one or more ionic species. Other workers have noted that CCK-like peptides produced an increase in input resistance of CA1 neurones (Boden and Hill, 1988b; Buckett and Saint, 1989) and suggested that block of a potassium current could be responsible, akin to that for CCK (and pentagastrin) actions on neurones in the rat lateral amygdala (Sugita et al., 1993). The proposal that CCK depolarizes hippocampal neurones by reducing outward current is supported by Brooks and Kelly (1985) who showed a decrease in input resistance of dentate gyrus neurones following CCK application. Two problems are associated with all of the experiments on hippocampal neurones outlined above, the lack of pharmacological identification of the CCK receptor and the fact that CCK responses from hippocampal neurones show profound desensitization. While the advent of novel CCK antagonists has helped with the characterization of the receptor the hippocampal preparation remains a poor model for CCK studies because of the limitations imposed by the desensitizing nature of the peptide response. More recently attempts have been made to identify the ionic conductances linked t o CCK-induced excitation of hypothalamic neurones. For example, selective antagonists have been used to identify a CCKB receptor whose activation leads to depolarization of neurones in the rat supraoptic nucleus (SON). Jarvis et al. (1992) reported that this CCK effect was mediated via
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activation of a nonselective cation conductance akin to that described by Petersen in rat pancreatic acinar cells (Petersen, 1987). Much work on the mechanism of action of C C K has centred around the CCKB receptor in the rat ventromedial hypothalamus or VMH. Several groups have confirmed the findings of Boden and Hill (1988a) who showed that CCK excited a large number (ca. 90%) of VMH neurones, and the vast majority of these cells were still influenced by pentagatrin in the presence of the CCKA antagonist devazepide confirming that the receptor involved was of the CCKB type (Boden et al., 1994). Neurones bathed in tetrodotoxin-containing, calcium-free solutions were still depolarized by CCK, suggesting that the effect was by an action at the postsynaptic membrane. There was also an inhibition of the GABA-mediated inhibitory postsynaptic potential (i.p.s.p.) when CCK was present in the bathing solution suggesting that the peptide can also reinforce the postsynaptically-induced excitation by reducing inhibitory transmitter release (Boden unpublished observations). Under voltageclamp conditions the effect of CCK was seen as a net inward current as a result of a reduction in a voltage-dependent outward current (Boden, 1991). Currentvoltage relationships showed this current to become activated as the neurone was depolarized from resting membrane potential and that CCK inhibited the activation of the current. However, CCK also reduced instantaneous inward current implying that at least two mutually reinforcing effects on separate currents were producing the pronounced excitation seen with the peptide under normal physiological conditions. The finding that CCK can influence several ion conductances is not unique to this peptide. For example, muscarinic agonists and substance P act on multiple conductances in rat locus coeruleus neurones (Shen and North, 1992a,b), and recently neurotensin has been shown to excite rate ventral tegmental neurones by reducing potassium and increasing sodium conductance (Jiang et al., 1994). One surprising feature, however, was the uncovering of a third effect of CCK under conditions when the voltage-dependent outward current was blocked using either carbachol or phorbol ester-12,13dibutyrate(PdBu) which is known to activate protein kinase C (Nishizuka, 1984). Treatment of CCK-sensitive VMH neurones with either of these agents produced effects akin to those seen when the peptide itself was applied but application of CCK during the response to PdBu (or carbachol) now produced an outward current. Under current-clamp conditions with no tetrodotoxin present this was seen as a membrane hyperpolarization leading to a reduction in the PdBu induced increase in firing of the cell. Thus, it appears that, under normal conditions, CCK acts by inhibition of two separate outward currents to increase neuronal excitability. Under conditions of increased neuronal excitation when the CCKsensitive current is blocked then the peptide activates an outward current. Activation of two opposing currents by one peptide has been reported previously (e.g., Bradykinin; Higashida and Brown, 1986). Attempts to prevent the excitatory action of CCK in rat V M H by pre-treatment with pertussis toxin (PTX) were unsuccessful (Boden, 1991). Taken together the data for CCK acting at CCKB receptors on neurones in the rat V M H suggest the involvement of a PTX insensitive G-protein linked to multiple ion conductances. Some of the effects of
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CCK can be mimicked by activators of protein kinase C but the involvement of a PKC step in the cascade of events following CCK binding to CCKB receptors awaits to be confirmed.
CCKA Receptors In the rat dorsal raphe CCK excites a subpopulation of 5-HT neurones (Boden et al., 1991), which differ from those responsive to the neuropeptide bombesin (Pinnock and Woodruff, 1991), via activation of a CCKA receptor. Experiments have been undertaken to see whether the transduction mechanism linked to this CCK receptor type is similar to either that found in the periphery. Unfortunately in peripheral assays for CCK, the receptor type and ionic mecahanism both appar to be species dependent. For example, in the rat and mouse acinar cell preparation the depolarizing effects of secretagogues can be seen with a rank order corresponding to the presence of a mixture of CCKB/gastrin and CCKA receptors (Iwatsuki et al., 1977) and CCK depolarizes pancreatic acinar cells by increasing calcium to activate a calcium-dependent nonselective monovalent cation channel. In the guinea pig (and probably human) CCK evokes amylase secretion, with a profile corresponding to action at a CCKA receptor only (Jensen et al., 1989), by increasing intracellular calcium to activate a calcium- and voltage-dependent potassium channel (Petersen, 1987; Petersen and Findtay, 1987). Voltage-clamp experiments in brain slice preparations have revealed that activation of the CCKA receptor reduces a potassium current to excite raphe neurones (Boden and Woodruff, in preparation). The potassium current involved is not dependent on extracellular calcium, since the inward current produced by CCK was not affected by removal of calcium ions or by the calcium-dependent potassium channel blockers, charybdotoxin (100 nM), and apamin (100nM). The response was unaffected by blockers of the voltage-dependent potassium current IA(dendrotoxin 100-300nM) or inward rectifier currents (rubidium, 5mM; caesium, 2 mM) but was blocked by barium (1-2 mM). In the rat midbrain, dopaminergic neurones of the substantia nigra zona compacta are excited by CCK, an effect which has been shown to be linked to activation of a nonselective cation conductance (Wu and Wang, 1994a). The CCK response was not blocked by selective CCKB antagonists suggesting that only CCKA receptors are involved. Further to this the same authors have shown that PTX pre-treatment does not affect the CCK response which agrees with earlier work demonstrating the same lack of effect of PTX on CCKB induced responses. The similarity between the transduction processes linked to CCKA and CCKB receptors is clear since both involve a PTX-insensitive G-protein. Wu and Wang (1994b) have also shown that the CCKA-mediated excitation of dopamine neurones is not blocked by intracellular injection of protein kinase C inhibitors such as PKC (19-31) and staurosporine but is blocked by heparin and intracellular calcium chelation, implying that release of intracellular calcium by inositol 1,4,5-trisphosphate is required for the CCKA-mediated response. However, whether CCK activates a calcium-dependent cation conductance by this
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route or whether the block of the CCK response is merely a consequence of loss of cation conductance following intracellular calcium depletion is not known. The above studies of transduction mechanisms linked to CCKA receptor activation provide no evidence as yet that this type of CCK receptor links to more than one type of ionic conductance in the mammalian brain. The summary table 3 below gives the known information on signaling mechanisms linked to pharmacologically identified CCK receptors in rat brain.
Brain region
Receptor
G-protein
VMH
CCKB
PTX-insensitive
CA1 hippocamus Lateral amygdala SON Dorsal raphe Substantia nigra
CCKB CCK8 CCK~
not known not known not known not known PTX-insensitive
Ion channel (transducer) ~, K + (pKC?) TK ÷
CCKA CCKA
~ K+ ~ K+ T Na+/K+ ~ K+ ~ Na÷/K ÷ (IP3?)
MOLECULAR BIOLOGY OF CCK RECEPTORS Pharmacological data have suggested that there may be as many as four different receptor types: C C K A , CCKB, gastrin, and CG-4 receptors preferring the COOH-terminal tetrapeptide common to CCK and gastrin, CG-4 (Menozzi et al., 1991). However molecular cloning techniques have only revealed two different receptor types. Many of the pharmacological differences observed may be explained by the existence of multiple affinity states and multiple modes of G-protein coupling. There are also considerable differences in antagonist selectivity and affinity that arise from variations in receptor sequence between species.
Cloning of
CCK A
and CCKn receptors
The C C K A receptor ( C C K A R ) w a s first isolated and purified from rat pancreatic tissue (Wank et al., 1992a). The receptor was purified by a variety of chromatographic steps and protein sequence data obtained from a number of CNBr and Lys-C peptides. Oligonucleotide primers were synthesized based on sequence obtained from two of the peptides and used to amplify a 527-bp product from cDNA of reverse transcribed rat pancreas mRNA by the polymerase chain reaction (PCR). This product was then used to screen a rat pancreatic cDNA .sAbbreviations: VMH = ventromedial hypothalamus, SON = supraoptic nucleus, K ÷ = potassium conductance, Na*/K" =nonselective cation conductance, pKC=protein kinase C, IP3=1,4,5 inositol triphosphate.
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library. All positive hybridising clones obtained contained the 5' untranslated region and most of the coding region. However the 3' end of the coding region was only obtained after using a variety of PCR methods. The full length eDNA encoded a 444 amino acid protein and was found to be a member of the G-protein coupled receptor family (Strader et aL, 1994). At the same time, the gastrin receptor was being cloned from canine parietal cells by a group from Boston using an expression cloning method (Kopin et al., 1992). R N A was extracted from a 95% pure population of parietal cells and reverse transcribed into eDNA. DNA greater than 1.5 kb was ligated into a mammalian expression vector (pcDNA) and two million primary recombinants in pools of 3000-10,000 were prepared by transformation of E.coli. D N A from each pool was then used to transfect COS-7 cells and positive pools identified by incubation with 125I labeled CCK analogues. The positive pool was then sequentially divided until a single positive clone was obtained. The sequence of the clone encoded a 453 amino acid protein. Tissue distribution was examined by high stringency Northern blot analysis of various adult canine tissues and proved to be consistent with CCKB receptor distribution. Therefore, it was proposed that the gastrin receptor and the CCKB receptor were identical. Pharmacological examination of COS-7 cells transfected with cloned the receptor demonstrated the binding specificity typical of gastrin/CCK~ receptors. Following these two initial reports there has been a plethora of further cloning papers from various different species and from various tissues, all suggesting that there are only two genes for the different receptor types. Indeed, Southern blot hybridization experiments suggests that there is only a single gene that encodes the gastrin and CCKB receptor (Lee et al., 1993; de Weerth et al., 1993a). The receptors sequenced to date are summarized in Table I. In general sequence homology is approximately 90-97% across species and around 50% across receptor type. Multiple sequence alignment of the two receptor types (Figs t and 2) highlights the sequence conservation across species. It should be noted that the rat CCKA receptor has a 15 amino acid longer N-terminus than the other cloned CCKA receptors. This is due to an additional ATG initiation codon upstream and in frame of the site where the other species begin. It is not known whether the rat CCKA receptor begins at the same position as the other receptors or with the additional amino acids. Recently a group has isolated the gene for the human CCKB receptor and found evidence for alternative splicing (Song et al., 1993). The gene was found to contain four introns, with the possibility of differential splicing at the end of the fourth exon. The two possible isoforms formed coded for 452 and 447 amino acid proteins. The shorter form sequence is the same as those previously reported, which are all eDNA clones (Pisegna et al., 1992; Lee et aL, 1993; Ito et al., 1993). The longer form sequence has an insert with the sequence Gly-Gly-Ala-Gly-Pro present after Pro-270. This sequence is present in the rat, guinea pig, canine and Mastomys sequences with a high level of conservation (Fig. 3). The existence of both splice variants was confirmed by PCR analysis of stomach m R N A (Song et al., 1993). The position of the splice variant is in the third cytoplasmic loop (see Fig. 4 for schematic representation of the human CCK~ receptor), a region
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Table I. CCK Receptors Cloned to Date Species (and source of clone) CCKA receptors Rat (pancreas) Human (gallbladder) Human (genomic) Guinea pig (gallbladder, pancreas) Rabbit CCKBreceptors Dog (parietal) Rat (brain and AR42J cells) Human (brain, stomach) Guinea pig (gallbladder) Mastomys(ECL tumor cell) Rabbit (genomic)
Size (amino acids) 444 428
Reference
428 430
Wank et al., 1992a de Weerth et al., 1993a, Ulrich et al., 1993 Pisegna et aL, 1994 de Weerth et aL, 1993b
427
Reuben et al., 1994
453 452
453
Kopin et al., 1992 Wank et al., 1992b, Jagerschmidt et al., 1994 Pisegna et al., 1992 Lee et aL, 1993, Ito et aL, 1993 de Weerth et al., 1993c
450
Nakata et al., 1992
452
Blandizzi et aL, 1994
447
known to be important for G-protein coupling (Ostrowski et al., 1992). Alternative splicing in this loop region of the D2 dopamine receptor has also been described and may affect receptor coupling (Montmayeur and Borrelli, 1991; Liu e t a l . , 1992). Therefore the two receptor isoforms may contribute to functional differences in CCKB receptor mediated signal transduction. C o m m o n Structural Features
In common with other members of the family of G-protein linked receptors the CCK receptor proteins are characterized by seven hydrophobic stretches which are predicted to form transmembrane a-helices. Figure 4 shows a schematic representation of the human brain CCKB receptor and is used to illustrate a number of common structural features. Most members of the G-protein coupled receptor superfamily are glycoproteins and are usually modified by N-linked glycosytation, with the consensus sequence Asn-Xxx-Ser/Thr. Glycosylation generally occurs in the N-terminal region before the first transmembrane region, but there are reports of other regions being glycosylated. The human CCKB receptor has three N-link glycosylation sites (residues 7, 30, and 36). The role of this general pattern of gylcosylation is not fully understood. Site directed mutagensis and biochemical analysis of several members of the receptor superfamity have shown that it is not required for ligand-binding or functional activity, but is probably required for receptor trafficking and expression on the cell surface (Fliesler and Basinger, 1985; Rands e t a l . , 1990). There are also a number of highly conserved residues that appear to be important for receptor structure. These include two highly conserved cysteine
I ...........
I ..........
5 ...........
I
7 .......
Fig. 1. S e q u e n c e alignment of cloned C C K , receptors. T r a n s m e m b r a n e regions indicated by dashed lines above the alignment. Rat-B = rat CCKB receptor s e q u e n c e , Mast-B = M a s t o m y s CCKr~ receptor sequence, Gpig-B = guinea pig CCKB receptor sequence, H u m - B = h u m a n CCKB receptor s e q u e n c e , D o g - B = canine CCKB receptor sequence, Rab-B = rabbit CCKB receptor sequence. See Table I for references. Note: H u m a n s e q u e n c e s h o w n is the short form isolated from brain.
..... [ MHRRFRQACLETCARCCPRPPRARPRALPDEDPPTPSIASLSRLSYTTISTLGPG MHRRFRQACLDTCARCCPRPPRARPRPLPEEDPPTPSIASLSRLSYTTISTLGPG MHRRFRQACLDTC;LRCCPRPPRARPQPLPDEDPPTPSIASLSRLSYTTISTLGPG MHRRFRQACLDTCARCCPRPPRARPRPLPDEDPPTPSIASLSRLSYTTISTLGPG MHRRFRQACLETCARCCPRPPRARPRPLPDEDPPTPSIASLSRLSYTTISTLGPG MHRRFRQACLDTCARCCPRPPRARPRPLPDEDPPTPSIASLSRLSYTTISTLGPG
i ............
Hum-B Gpig-B Rat-B Mast-B Dog-B Rab-B
I
VQLPRSRP~LLELSALAASTPAPGPGPRPTQAKLLA~KR~RMLLVI~LFFLC~LPVYSANTWRAFDGPGAHRALSGAPISFIHLLSYAsACVNPL~YcF VQLPRSR.~LEMTTLTTPTPGPVPGPRPNQ~LLAKKR~RMLLVIVLLFFLCwLPVYSVNTWRAFDGPGAQRALSGAPISFIHLLSYVSACVNPLVYcF VQLPRSR.•LEMTTLTTPTPGPGLAS.ANQAKLLAKKR•RMLLVIVLLFFLCWLPIYSANTWCAFDGPGAHRALSGAPISFIHLLSYASACVNPLvYCF VQLPRSRQTLELS;LLTAPTPGPGGGPRPYQAKLLAKKR•RMLL•I•LFFLCWLPLYSANTNRAFDSSGAHRALSGAPISFIHLLSYASACVNPLVY•F VQLPRSRPALELS~LTAPIsGPGPGPRPAQAKLLAKKR~RMLLVIV~LFFMCWLPVYSANTWRAFDGPGAHRALSGAPISFIHLLSYASACVNPLVYCF
I ........... 6 ........... VQLPRSRPALELTALT•.APGPGSGSRPTQAKLLAKKRwRMLL•I•LFFLCWLPVYSANTwRAFDGPGAHRALSGAPISFIHLLSYASAc•NPL•YCF
PRVLQCVHRWPSARVRQTNSVLLLLLLFFIPGVVMAVAYGLISRELYLGLRFDGDSDSDSQSRVRNQGGLP ..... GAVHQNGRCRPETGAVGEDSDGCY PRVLQCMHRwPSARVRQTWSvLLLLLLFFVPGVVMAVAYGLISRELYLGLHFDGDADSESQ•RVRGPGGLSG.SAPGPAHQNGRCRPESGLSGEDSDGCY PRVLQCMHRw•SARVQQTWSVLLLLLLFFIPGwIAVAYGLISRELYLGLHFDGENDSETQSRARNQGGLPGGAAPGPVHQNGGCRPVTSVAGEDSDGCC PRVLQCMHRWPSARVRQTWSvLLLMLLFFIPGVVMAVAYGLISRELYLGLRFDGDNDSDTQSR•RNQGGLPGGTAPGPvHQNGGCRHVT.VAGEDNDGCY ARALQCVHRwPSAR•RQTWSVLLLLLLF•VPGVVMAvAYGLISRELYLGLRFDEDSDSE.•SR•RSQGGLRGGAGPGPAPPNGSCRPEGGLAGEDGDGCY PR~LQCVHRWPSARVRQTWSvLLLLLLFFVPGV~MAVAYGLISRELYLGLRFDSDSDSESQSRVRGQGGLPGGAAPGPVHQNGRCAPEAGLAGEDGDGCY
Hum-B Gpig-B Rat-B Mast-B Dog-B Rab-B
Hum-B Gpig-B Rat-B Mast-B Dog-B Rab-B
2........ I I.......... 3.......... I I......... 4.......... I LLLAVACMPFTLLPNLMGTF•FGT••CKA•SYLMG•SVS•STLSL•AIALER•SAICRPLQARVwQTRSHAARVI•ATWLLSGLLMVPYPVYT•QP•VG LLLAVACMPFTLLPNLMGTFIFGTVICKAV•YLMGVSVSVSTLSLVAIALERY•AICRPLQARVwQTRSHAARVILATWLL•GLLMVPYPVYTAVQP•VG LLLAVACMPFTLLPNLMGTFIFGTVICKAI•YLMG•SVSVSTLNLVAIALERYSAICRPLQARVwQTRSHAARVILATWLLSGLLMVPYPVYTMVQP.VG LLLAVACMPFTLLPNLMGTFIFGTVIcKAVSYLMGVSVSVSTLNLVAIALERYSAICR•LQARVwQTRSHAARVILATWLLSGLLMVPYPVYT•QP.VG LLLAVACMPFTLLPNLMGTFIFGTVvCKAVSYLMGVSVSVsTLSLVAIALERYSAICRPLQARVwQTRSHAARVIIAT•MLSGLLMVPYPVYTAVQPAGG LLLAVACMPFTLLPNLMGTFIFGTVICKAVSYLMGVSVSVSTLSLVAIALERYSAICR•LQ;LRVwQTRSHAARVILAT•LLSGLLMVPYP•YTAVQP.VG
I
Hum-B Gpig-B Rat-B Mast-B Dog-B Rab-B
1 ............
MELLKLNRSLQGPGPGSGAPLCRPAGPLLNSSGAGNVSCETPRIRGAGTRELELAIRVTLYAVIFLMSVGGNVLIIWLGL•RRLRTVTNAFLLSLAV•D MELLKLNRSVQGPGPGSGSSLCRPGvSLLNSSSAGNLScDPPRIRGTGTRELEMAIRITLYAVIFLMSVGGNVLIIWLGLSRRLRTVTNAFLLSLAVSD MELLKLNSSVQGPGPGSGSSLCHPGVSLLNSSSAGNLSCEPPRIRGTGTRELELAIRITLYAVIFLM•IGGNMLIIwLGLSRRLRT•TNAFLLSLAVSD MELLKLNRSAQGSGAGP•ASLCRAGGALLNSSGAGNLSCEPPRLRGAGTRELELAIRVTLYAvIFLM•VGGNVLII•LGLSRRLRTvTNAFLL•LAVSD MELVKLNR•VQGSGP..VASLCRPGGPLLNNSGTGNLSCEPPRIRGAGTRELELAI•VTLYAVIFLMSVGGNILIIwLGLSRRLRTVTNAFLLSLAVSD
I .......... MELLKLNRSvQGTGPGPGASLCRPGAPLLNSSSVGNL•CEPPRIRGAGTRELELAIRITLYAVIFLMSVGGNMLII•LGLSRRLRTVTNAFLLSLAV•D
Hum-B spig-S Rat-B Mast-B Dog-B Rab-B
Qt~
g
o
t~
I. . . . . . . . . . .
1 . . . . . . . . . . .
I
I
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I
I. . . . . . . . . .
3. . . . . . . . . .
I
I..........
4. . . . . . . . .
I
5 ...........
l
J ...........
7 .............
i
Fig. 2. Sequence alignment of cloned CCKA receptors. T r a n s m e m b r a n e regions indicated by dashed lines above the alignment. H u m - A = human CCKA receptor sequence, Gpig-A = guinea pig CCKA receptor sequence, Rab-A = rabbit CCKA receptor sequence, Rat-A = rat CCKA receptor sequence.
TFPCCPNPGPPGARGEVGEEEEGGTTGASLSRFSYSHMSASVPPQ TFPCCPNPGTPGVRGEMGEEEEGRTTGASLSRYSYSHMSTSAPPP TFPCCPNPGPPGPRAEAGEEEEGRTTKASLSRYSYSHMSASAHAS TFPCCPNPGPPGVRGEVGEEEDGRTIRALLSRYSYSHMSTSAPPP
I
Hum-A Gp-A Rab-A Rat-A
6 ...........
STGSSS..RANRIRSNSSAANLMAKKRVIRMLIvIVvLFFLCwMPIFSANAWRAYDTASAERRLSGTPISFILLLSYTSSCVNPIIYC~l~NKRFRLGFMA SPSSSGSNRINRIRSSSSTANLMAKKRVIRMLIVIVvLFFLCWM•IFSANAwRAYDTVSAERHLSGTPISFILLLSYTSSCVNPIIYCFMNKRFRLGFMA SGGGGG••RV•R•HS•SSAAALMAKKRVIRMLMV•wLFFLcWMP•FSANA•RAYDTVSAERRLSGTPIS•ILLLSYTSSCVNPIIYCFMNKRFRLGFMA SSGSGGS•RLNRIRSSSSAANLIAKKRVIRMLIVIwLFFLcWMPIFSANAwKAYDTVSAEKHLSGT•ISFILLLSYTSSC•NPIIYCFMNKRFRLG•MA
I ...........
TKNNNQTANMcRFLL•NDVMQQSWHTFLLLILFLIPGIVMMVAYGLISLELYQGIKFEASQKKSAKERKPSTTSSGKYEDSDGCYLQKTRPPRKLELRQL T~NNQTGNMCRFLLPNDVMQQTWHTFLLLILFLIPGIVMMVAYGLISLELYQGIKFDAIQKKSAKERKTSTGSSGPMEDSDGcYLQKSRHPRKLELRQL TKTNNQTANMCRFLLPSDvMQQAWHTFLLLILFLIPGIVMMVAYGMISLELYQGIKFDASQKKS;d
