Structural difference of vasoactive intestinal peptide in two distinct membrane-mimicking environments.

Vasoactive intestinal peptide (VIP) is a 28-amino acid neuropeptide which belongs to a glucagon/secretin superfamily, the ligand of class II G protein-coupled receptors. Knowledge for the conformation of VIP bound to membrane is important because the receptor activation is initiated by membrane binding of VIP. We have previously observed that VIP-G (glycine-extended VIP) is unstructured in solution, as evidenced by the limited NMR chemical shift dispersion. In this study, we determined the three-dimensional structures of VIP-G in two distinct membrane-mimicking environments. Although these are basically similar structures composed of a disordered N-terminal region and a long α-helix, micelle-bound VIP-G has a curved α-helix. The side chains of residues Phe(6), Tyr(10), Leu(13), and Met(17) found at the concave face form a hydrophobic patch in the micelle-bound state. The structural differences in two distinct membrane-mimicking environments show that the micelle-bound VIP-G localized at the water-micelle boundary with these side chains toward micelle interior. In micelle-bound PACAP-38 (one of the glucagon/secretin superfamily peptide) structure, the identical hydrophobic residues form the micelle-binding interface. This result suggests that these residues play an important role for the membrane binding of VIP and PACAP.

Molecular mechanisms involved in vasoactive intestinal peptide receptor activation and regulation: current knowledge, similarities to and differences from the A family of G-protein-coupled receptors.

An actual paradigm for activation and regulation of the GPCR (G-protein-coupled receptors)/seven-transmembrane helix family of receptors essentially emerges from extensive studies of the largest family of receptors, the GPCR-A/rhodopsin family. The mechanisms regulating the GPCR-B family signal transduction are less precisely understood due in part to the lack of the conserved signatures of the GPCR-A family (E/DRY, NPXXY) and in part to the absence of a reliable receptor modelling, although some studies suggest that both families share similar features. Here, we try to highlight the current knowledge of the activation and the regulation of the VIP (vasoactive intestinal peptide) receptors, namely VPAC (VIP/pituitary adenylate cyclase-activating peptide receptor) 1 and 2. This includes search for amino acids involved in the stabilization of the receptor active conformation and in coupling to G-proteins, signalling pathways activated in response to VIP, agonist-dependent receptor down-regulation, phosphorylation and internalization as well as pharmacological consequences of receptor hetero-dimerization.

Novel analogs of VIP with multiple C-terminal domains.

The effect of multiplication of the N-terminal domain of vasoactive intestinal peptide (VIP) on the binding activity of the peptide was recently evaluated. A VIP analog with multiple N-terminal domains was found to be slightly more potent as compared to [Nle(17)]VIP towards VIP receptor type 1 (VPAC1)-related cAMP production. Here, the effect of multiplication of the C-terminal domain of VIP was evaluated with the aim of possibly amplifying peptide-receptor (VPAC1) binding and activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the C-terminal domain that was obtained by either a simple linear tandem extension or by a unique branching methodology. Results show that despite significant alterations in the C-terminal domain of VIP that is considered essential to induce potent receptor binding, few peptides demonstrated only slight reduction in receptor binding and activation in comparison to [Nle(17)]VIP. Furthermore, a specific branched VIP analog with multiple C-terminal domains was equipotent to [Nle(17)]VIP in the cAMP production assay. Therefore, it is concluded that the association between the VIP ligand to the VIP receptor could be tolerable to size increases in the C-terminal region of the VIP ligand and multiplication of the C-terminal does not increase activity.

Molecular cloning and functional expression of a VIP-specific receptor.

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.

Novel concepts of neuropeptide-based drug therapy: vasoactive intestinal polypeptide and its receptors.

Chronic inflammatory airway diseases such as bronchial asthma or chronic obstructive pulmonary disease (COPD) are major contributors to the global burden of disease. Although inflammatory cells play the central role in the pathogenesis of the diseases, recent observations indicate that also resident respiratory cells represent important targets for pulmonary drug development. Especially targeting airway neuromediators offers a possible mechanism by which respiratory diseases may be treated in the future. Among numerous peptide mediators such as tachykinins, calcitonin gene-related peptide, neurotrophins or opioids, vasoactive intestinal polypeptide (VIP) is one of the most abundant molecules found in the respiratory tract. In human airways, it influences many respiratory functions via the receptors VPAC1, VPAC2 and PAC1. VIP-expressing nerve fibers are present in the tracheobronchial smooth muscle layer, submucosal glands and in the walls of pulmonary and bronchial arteries and veins. Next to its strong bronchodilator effects, VIP potently relaxes pulmonary vessels, and plays a pivotal role in the mediation of immune mechanisms. A therapy utilizing the respiratory effects of VIP would offer potential benefits in the treatment of obstructive and inflammatory diseases and long acting VIP-based synthetic non-peptide compounds may represent a novel target for drug development.

Contribution of the carboxyl terminus of the VPAC1 receptor to agonist-induced receptor phosphorylation, internalization, and recycling.

When exposed to vasoactive intestinal peptide (VIP), the human wild type VPAC1 receptor expressed in Chinese hamster ovary (CHO) cells is rapidly phosphorylated, desensitized, and internalized in the endosomal compartment and is not re-expressed at the cell membrane within 2 h after agonist removal. The aims of the present work were first to correlate receptor phosphorylation level to internalization and recycling, measured by flow cytometry and in some cases by confocal microscopy using a monoclonal antibody that did not interfere with ligand binding, and second to identify the phosphorylated Ser/Thr residues. Combining receptor mutations and truncations allowed identification of Ser250 (in the second intracellular loop), Thr429, Ser435, Ser448 or Ser449, and Ser455 (all in the distal part of the C terminus) as candidates for VIP-stimulated phosphorylation. The effects of single mutations were not additive, suggesting alternative phosphorylation sites in mutated receptors. Replacement of all of the Ser/Thr residues in the carboxyl-terminal tail and truncation of the domain containing these residues completely inhibited VIP-stimulated phosphorylation and receptor internalization. There was, however, no direct correlation between receptor phosphorylation and internalization; in some truncated and mutated receptors, a 70% reduction in phosphorylation had little effect on internalization. In contrast to results obtained on the wild type and all of the mutated or truncated receptors that still underwent phosphorylation, internalization of the severely truncated receptor was reversed within 2 h of incubation in the absence of the agonist. Receptor recovery was blocked by monensin, an endosome inhibitor.

Effect of inactivating mutations on phosphorylation and internalization of the human VPAC2 receptor.

The VPAC(2) receptor, as all members of the G-protein-coupled receptor (GPCR)-B family, has two highly conserved motifs in the third intracellular (IC(3)) loop: a lysine and a leucine located at the amino-terminus and two basic residues separated by a leucine and an alanine at the carboxyl-terminus. This study evaluates the involvement of those conserved amino acid sequences in VPAC(2) signal transduction and regulation. The residues were mutated into alanine and mutants were expressed in Chinese hamster ovary (CHO) cells stably transfected with Galpha16 and aequorin. Mutation of L310 reduced efficacy of vasoactive intestinal polypeptide (VIP) to stimulate adenylate cyclase activity through Galphas coupling by 75%, without affecting VIP capability to stimulate an increase in [Ca(2+)](i) through Galpha16 coupling. Mutation of R325 and, to a lesser extend, K328 reduced VIP efficacy to stimulate [Ca(2+)](i) increase and VIP potency to stimulate adenylate cyclase. The combination of mutations of both amino- and carboxyl-terminus located conserved motifs of the IC(3) loop generates an inactive receptor with respect to [Ca(2+)](i) increase and adenylate cyclase activation, but also with respect to receptor phosphorylation and internalization that were indeed directly correlated with the potency of inactivation of the receptors. The amino-terminus of the VPAC(2) receptor IC(3) loop is thus involved in adenylate cyclase activation and the carboxyl-terminus of the IC(3) loop participates in both Galphas and Galpha16 coupling. The mutations studied also reduced both receptor phosphorylation and internalization in a manner that appeared directly linked to the alteration of Galphas and Galpha16 coupling.

Mutations in the carboxy-terminus of the third intracellular loop of the human recombinant VPAC1 receptor impair VIP-stimulated [Ca2+]i increase but not adenylate cyclase stimulation.

The vasoactive intestinal polypeptide (VIP) VPAC1 receptor is preferentially coupled to Galphas protein that stimulates adenylate cyclase activity and also to Galphaq and Galphai proteins that stimulate the inositol phosphate/calcium pathway. Previous studies indicated the importance of the third intracellular loop of the receptor for G protein coupling. By site-directed mutation of the human recombinant receptor expressed in Chinese hamster ovary cells, we identified two domains in this loop that contain clusters of basic residues conserved in most of the G-protein-coupled seven transmembrane domains receptors. We found that mutations in the proximal domain (K322) reduced the capability of VIP to increase adenylate cyclase activity without any change in the calcium response, whereas mutations in the distal part of the loop (R338, L339, R341) markedly reduced the calcium increase and Galphai coupling but only weakly the adenylate cyclase activity. Thus, the interaction of different G proteins with the VPAC1 receptor involves different receptor sub-domains.

Presence of a N-terminal signal peptide in class II G protein-coupled receptors: crucial role for expression of the human VPAC1 receptor.

The hVPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylyl cyclase activating peptide (PACAP) has an N-terminal signal peptide like all other class II G protein-coupled receptors (GPCRs). We determined the role of the signal peptide in expression of human VPAC1 receptor in transfected CHO cells. Three constructs were transfected: Flag30-hVPAC1, a receptor containing an inserted FLAG sequence between Ala30 and Ala31 and fused in the C-terminal position to GFP; Flag30-[delta1-30]-hVPAC1, the same construct as Flag30-hVPAC1 but lacking the 1-30 putative signal peptide (SP) sequence; Flag0-hVPAC1, a receptor containing an N-terminal FLAG sequence and fused in the C-terminal position to GFP. For each construct, we determined 125I-VIP binding, VIP-induced cAMP production, GFP fluorescence and indirect immunofluorescence on nonpermeabilized cells incubated with mouse monoclonal anti-Flag antibodies. The data were consistent with a crucial role of the signal peptide for expression of functional VPAC1 receptors at the cell surface and suggested that the signal peptide is cleaved during the translocation of the receptor to the plasma membrane, probably in the endoplasmic reticulum.

Ac His1 [D-Phe2, K15, R16, L27] VIP (3-7)/GRF (8-27)--a VPAC1 receptor antagonist--is an inverse agonist on two constitutively active truncated VPAC1 receptors.

C-terminally truncated human VPAC(1) receptors were constructed and stably transfected in Chinese hamster ovary (CHO) cells. Selected clones expressing comparable receptor densities were studied for ligand's binding properties, basal and stimulated adenylate cyclase activity. The wild-type (1-457) receptor served as reference. The binding properties of all the constructions were preserved. As judged by the intrinsic activity of the partial agonist Q(3)-VIP, the shortest receptors have a moderate impairment of the coupling efficacy to G(alpha s) protein. Cells expressing the VPAC(1) (1-436) and (1-441) truncated receptors had a two- to three-fold higher basal adenylate cyclase activity than those expressing the wild-type or the VPAC(1) (1-444), (1-433), (1-429), (1-421) and (1-398) receptor. The stimulatory effect of VIP and other agonist was preserved. This suggested that VPAC(1) (1-436) and (1-441) receptors had a constitutive activity. The selective VPAC(1) receptor antagonist Ac His(1) [D-Phe(2), K(15), R(16), L(27)] VIP (3-7)/GRF (8-27) reduced by 60% the basal activity with an EC(50) value of 3 nM comparable to its IC(50) value for binding. This agonist behaved thus like an inverse agonist on the constitutively active VPAC(1) receptors generated by C-terminal truncation and expressed in CHO cells.

A natural variant type II G protein-coupled receptor for vasoactive intestinal peptide with altered function.

The vasoactive intestinal peptide (VIP) and its G protein-coupled receptors VPAC1 and VPAC2 prominently mediate diverse physiological functions in the neural, endocrine, and immune systems. A deletion variant of mouse VPAC2 has been identified in immune cells that lacks amino acids 367-380 at the carboxyl-terminal end of the seventh transmembrane domain. When expressed at equivalent levels in a human Jurkat T cell line, which has very low endogenous expression of human VPAC1 and VPAC2, wild-type and deletion-variant VPAC2 bound the same amount of 125I-VIP with similar affinity. Unlike wild-type VPAC2, however, deletion-variant VPAC2 did not transduce VIP-elicited increases in intracellular concentration of cyclic AMP, chemotaxis, or suppression of generation of interleukin-2. Natural deletion of part of the last transmembrane domain of VPAC2 thus abrogates signaling functions without apparent alterations of expression or ligand binding.

Diffuse pharmacophoric domains of vasoactive intestinal peptide (VIP) and further insights into the interaction of VIP with the N-terminal ectodomain of human VPAC1 receptor by photoaffinity labeling with [Bpa6]-VIP.

The widespread 28-amino acid neuropeptide vasoactive intestinal peptide (VIP) exerts its many biological effects through interaction with serpentine class II G protein-coupled receptors named VPAC receptors. We previously provided evidence for a physical contact between the side chain at position 22 of VIP and the N-terminal ectodomain of the hVPAC1 receptor (Tan, Y. V., Couvineau, A., Van Rampelbergh, J., and Laburthe, M. (2003) J. Biol. Chem. 278, 36531-36536). We explored here the contact site between hVPAC1 receptor and the side chain at position 6 of VIP by photoaffinity labeling. The photoreactive para-benzoyl-l-Phe (Bpa) was substituted for Phe(6) in VIP resulting in [Bpa(6)]-VIP, which was shown to be a hVPAC1 receptor agonist in Chinese hamster ovary cells stably expressing the recombinant receptor. After obtaining the covalent (125)I-[Bpa(6)-VIP].hVPAC1 receptor complex, it was sequentially cleaved by cyanogen bromide, peptide N-glycosidase F, endopeptidase Glu-C, and trypsin, and the cleavage products were analyzed by electrophoresis. The data demonstrated that (125)I-[Bpa(6)-VIP] were covalently attached to the short 104-108 fragment within the N-terminal ectodomain of the receptor. The data were confirmed by creation of a receptor mutant with new CNBr cleavage site. In a three-dimensional model of the receptor N-terminal ectodomain, this fragment was located on one edge of the putative VIP-binding groove and was adjacent to the fragment covalently attached to the side chain at position 22 of VIP. Altogether these data showed that the central part of VIP, at least between Phe(6) and Tyr(22), interacts with the N-terminal ectodomain of the hVPAC1 receptor.

Serine 447 in the carboxyl tail of human VPAC1 receptor is crucial for agonist-induced desensitization but not internalization of the receptor.

The VPAC1 receptor for vasoactive intestinal peptide (VIP) belongs to the class II family of G protein-coupled receptors and is coupled to Gs protein/adenylyl cyclase. We assessed whether 10 different Ser/Thr residues in human VPAC1 receptor intracellular domains play a role in the process of VIP-induced desensitization/internalization by performing a site-directed mutagenesis study. The Ser/Thr residues mutated to Ala include potential G protein-coupled receptor kinase, protein kinase A and protein kinase C targets that are of particular interest for VPAC1 receptor desensitization. The data show that when Chinese hamster ovary cells expressing wild-type receptors were pretreated for 5 min with VIP (50 nM), receptor desensitization occurred with a 10-fold right shift of the ED50 for adenylyl cyclase activation. When the construct with the widest span of mutations was studied, there was no longer any short-term desensitization. By using constructs with fewer and fewer mutations, we identified Ser447 in the C-terminal tail to be crucial for rapid desensitization. We also showed that Ser447 plays an essential role for VIP-induced VPAC1 phosphorylation in Chinese hamster ovary cells. Furthermore, we demonstrated that none of the mutated Ser/Thr residues was involved in down-regulation after a 12-h treatment of cells with 50 nM VIP. Neither were they involved in VIP and VIP-induced receptor internalization as shown using a novel fluorescein-tagged VIP and VPAC1 receptor bearing a Flag epitope in the N-terminal domain and a green fluorescent protein at the C terminus. We conclude that Ser447, a likely G protein-coupled receptor kinase target, is crucial for VIP-induced phosphorylation and rapid desensitization of VPAC1 receptor.

Photoaffinity labeling demonstrates physical contact between vasoactive intestinal peptide and the N-terminal ectodomain of the human VPAC1 receptor.

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide whose actions are mediated by VPAC receptors belonging to class II G protein-coupled receptors. To identify contact sites between VIP and its VPAC1 receptor, an analog of VIP substituted with a photoreactive para-benzoyl-l-Phe (Bpa) at position 22 has been synthesized and evaluated in Chinese hamster ovary cells stably expressing the recombinant human receptor. Bpa22-VIP and native VIP are equipotent in stimulating adenylyl cyclase activity in cell membranes. Cyanogen bromide cleavage of the covalent 125I-[Bpa22-VIP]-hVPAC1R complex yielded a single labeled fragment of 30 kDa that shifted to 11 after deglycosylation, most consistent with the 67-137 fragment of the receptor N-terminal ectodomain. Further cleavage of this fragment with V8 endoproteinase and creation of receptor mutants with new CNBr cleavage sites (XàMet), demonstrated that 125I-[Bpa22-VIP] was covalently attached to the short receptor 109-120 fragment (GWTHLEPGPYPI). In a three-dimensional model of the receptor N-terminal ectodomain, this fragment is located on one edge of the putative VIP binding groove and encompasses several amino acids previously shown to be crucial for VIP binding (reviewed in Laburthe, M., Couvineau, A., and Marie, J. C. (2002) Receptors Channels 8, 137-153). Our data provide the first direct evidence for a physical contact between VIP and the N-terminal ectodomain of the hVPAC1 receptor.

Evidence for a direct interaction between the Thr11 residue of vasoactive intestinal polypeptide and Tyr184 located in the first extracellular loop of the VPAC2 receptor.

We developed previously VPAC(1) [vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide (PACAP) receptor]>VPAC(2) receptor selective ligands. Replacement of the VIP-Thr(11) by an Arg(11) in these ligands contributed to their selectivity: Arg(11)-VIP had a 200-fold lower affinity when compared with VIP at VPAC(2) receptors as opposed to 3- to 5-fold higher affinity at VPAC(1) receptors. Comparison of the binding and functional properties of related VIP analogues suggested that the VPAC(1) selectivity of Arg(11)-VIP was due to the loss of a hydrogen bond between the hydroxy group of Thr residue and the VPAC(2) receptor, steric hindrance between the Arg side chain and the VPAC(2) receptor and charge attraction by the VPAC(1) receptor. Comparison of the ability of VIP analogues to activate adenylate cyclase through chimaeric VPAC(1)/VPAC(2) and VPAC(2)/VPAC(1) receptors indicated that the first extracellular receptor loop carried most of the VPAC(2) receptors' ability to discriminate VIP from Arg(11)-VIP. Based on results obtained for a truncated VPAC(2) receptor and the closely related PACAP-preferring receptor (PAC(1)) and secretin receptors, we hypothesized that Thr(11) interacted with the VPAC(2) receptor Tyr(184) (similar to the VPAC(1) receptor Phe(200) residue). The Y184F (Tyr(184)-->Phe) VPAC(2) mutant lost the ability to discriminate VIP from Val(11)-VIP, and the F200Y VPAC(1) mutant acquired the ability to discriminate the natural peptide from Val(11)-VIP. These results support the hypothesis that the hydroxy group of the native VIP-Thr(11) side chain can indeed form a hydrogen bond with the Tyr side chain in the VPAC(2) receptor.

Elucidation of the vasoactive intestinal peptide pharmacophore for VPAC(2) receptors in human and rat and comparison to the pharmacophore for VPAC(1) receptors.

Vasoactive intestinal peptide (VIP) functions as a neurotransmitter involved in a number of physiological and pathological conditions. The actions of VIP are mediated through VPAC(1) and VPAC(2). In contrast to VPAC(1), which has been extensively studied, little is known about the pharmacology of VPAC(2). In this study we investigated the VIP pharmacophore for VPAC(2) by using alanine and D-amino acid scanning. We found significant species differences, and the human VPAC(2) (hVPAC(2)) expressed in Chinese hamster ovary (CHO) cells, which have been used in previous studies, differed significantly from the native hVPAC(2) in Sup T(1) cells and hVPAC(2) expressed in PANC1 cells. There was a close agreement between binding affinities and potencies for VPAC(2) activation. The amino acids whose backbone or side chain orientations were most important for high affinity potency are Asp(3), Phe(6), Thr(7), Tyr(10), Arg(12), Tyr(22), and Leu(23), whereas the side chains of Ser(2), Asp(8), Asn(9), Gln(16), Val(19), Lys(20), Lys(21), Asn(24), and Ser(25) are not essential. Comparison of the VIP pharmacophore between hVPAC(1) and hVPAC(2) demonstrated that the side chains of Thr(7), Tyr(10), Thr(11), and Tyr(22) were much more critical for high affinity for the hVPAC(2) than the hVPAC(1). In contrast, the orientation of the side chain of Asn(24) was more important for high affinity for the hVPAC(1). This study shows that in assessing the pharmacophore of VIP analogs for the VPAC(2), important species differences need to be considered as well as the expression system used. These results of our study should be useful for designing VPAC subtype-selective analogs, simplified analogs, and possibly metabolically stable analogs.

Molecular pharmacology and structure of VPAC Receptors for VIP and PACAP.

VIP and PACAP are two prominent neuropeptides which share two common G protein-coupled receptors VPAC1 and VPAC2 while PACAP has an additional specific receptor PAC1. This paper reviews the present knowledge regarding three aspects of VPAC receptors including: (i). receptor specificity towards natural VIP-related peptides and pharmacology of synthetic agonists or antagonists; (ii). receptor signaling; (iii). molecular basis of ligand-receptor interaction as determined by site-directed mutagenesis, construction of receptor chimeras and structural modeling.

A small sequence in the third intracellular loop of the VPAC(1) receptor is responsible for its efficient coupling to the calcium effector.

The stimulatory effect of vasoactive intestinal peptide (VIP) on the intracellular calcium concentration ([Ca(2+)](i)) has been investigated in Chinese hamster ovary cells stably transfected with the reporter gene aequorin, and expressing human VPAC(1), VPAC(2), chimaeric VPAC(1)/VPAC(2) or mutated receptors. The VIP-induced increase in [Ca(2+)](i) was linearly correlated with receptor density, and was higher in cells expressing VPAC(1) receptors than in cells expressing a similar density of VPAC(2) receptors. The study was performed to establish the receptor sequence responsible for this difference. VPAC(1)/VPAC(2) chimaeric receptors were first used for broad positioning: those receptors having the third intracellular loop (IC3) of the VPAC(1) or the VPAC(2) receptor behaved, in this respect, phenotypically like VPAC(1) and VPAC(2) receptors respectively. Replacement in the VPAC(2) receptor of the sequence comprising residues 315-318 (VGGN) within IC3 by its VPAC(1) receptor counterpart (residues 328-331; IRKS) and the introduction of VGGN instead of IRKS into VPAC(1) was sufficient to mimic VPAC(1) and VPAC(2) receptor characteristics respectively. Thus a small sequence in the IC3 domain of the VPAC(1) receptor is responsible for the efficient agonist-stimulated increase in [Ca(2+)](i).

Two tyrosine residues in the first transmembrane helix of the human vasoactive intestinal peptide receptors play a role in supporting the active conformation.

1: We investigated the human vasoactive intestinal polypeptide (VIP) receptors VPAC(1) and VPAC(2) mutated at conserved tyrosine residues in the first transmembrane helix (VPAC(1) receptor Y146A and Y150A and VPAC(2) receptor Y130A and Y134A). 2: [(125)I]-Acetyl-His(1) [D-Phe(2), K(15), R(16), L(27)]-VIP (1-7)/GRF (8-27) (referred to as [(125)I]-VPAC(1) antagonist) labelled VPAC(1) binding sites, that displayed high and low affinities for VIP (IC(50) values and per cent of high affinity binding sites: wild-type, 1 nM (57+/-9%) and 160 nM; Y146A, 30 nM (40+/-8%) and 800 nM; Y150A, 4 nM (27+/-8%) and 300 nM). [R(16)]-VIP behaved as a "super agonist" at both mutated VPAC(1) receptors and the efficacies of VIP analogues modified in positions 1, 3 and 6 were significantly decreased. 3: VIP was less potent at the Y130A and Y134A mutated VPAC(2) receptors (EC(50) 200 and 400 nM, respectively) than at the wild-type VPAC(2) receptor (EC(50) 7 nM). Furthermore, [hexanoyl-His(1)]-VIP behaved as a "super agonist" at the two mutated VPAC(2) receptors, and VIP analogues modified in positions 1, 3 and 6 were less potent and efficient at the mutated than at wild-type VPAC(2) receptors. However, the Y130A and Y134A mutants could not be studied in binding assays. 4: Our results suggest that the conserved tyrosine residues do not interact directly with the VIP His(1), Asp(3) or Phe(6) residues (that are necessary for receptor activation), but stabilize the correct active receptor conformation.

Human VPAC1 receptor selectivity filter. Identification of a critical domain for restricting secretin binding.

The human VPAC1 receptor for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase activating peptide (PACAP) belongs to the class II family of G protein coupled receptors with seven transmembrane segments. It recognizes several VIP-related peptides and displays a very low affinity for secretin despite >70% homology between VIP and secretin. Conversely, the human secretin receptor has high affinity for secretin but low affinity for VIP. We took advantage of this reversed selectivity to identify a domain of the VPAC1 receptor responsible for selectivity toward secretin by constructing human VPAC1-secretin receptor chimeras. A first set of chimeras consisted of exchanging the entire N-terminal ectodomain or large parts of this domain. They were constructed by overlap PCR, transfected in COS-7 cells, and their ligand selectivity, expressed as the ratio of EC(50) for secretin/EC(50) for VIP (referred to as S/V), in stimulating cAMP production was measured. Two very informative chimeras respectively referred to as S144V and S123V were obtained by replacing the entire ectodomain or only the first 123 amino acids of the VPAC1 receptor by the corresponding sequences of the secretin receptor. Whereas S144V no longer discriminated between VIP and secretin (S/V = 1.2), S123V discriminated between the two peptides (S/V = 300) in the same manner as the wild-type VPAC1 receptor. The motif responsible for discrimination was determined by introducing small blocks or individual amino acids of secretin receptor in the 123-144 sequence of the S123V chimera. The data obtained from 14 new chimeras sustained that two nonadjacent pairs of amino acids, Gln(135) Thr(136) and Gly(140) Ser(141) in the C-terminal end of the N-terminal VPAC1 receptor ectodomain constitute a selective filter that strongly restricts access of secretin to the VPAC1 receptor.