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PubMed1998

Stable expression of the mouse levocabastine-sensitive neurotensin receptor in HEK 293 cell line: binding properties, photoaffinity labeling, and internalization mechanism.

The recently cloned new subtype of G protein-coupled neurotensin receptor (NTRL) was stably expressed in the HEK 293 cell line in order to investigate its binding and internalization properties. The expressed receptor exhibited the typical binding characteristics of the low affinity, levocabastine-sensitive binding site previously described in rat and mouse brain and was detected as a protein with an apparent MW of 45 kDa by photoaffinity labeling. Although intracellular modulation of adenylate cyclase, guanylate cyclase and phospholipase C was not detected after application of neurotensin or levocabastine on NTRL-transfected cells, this receptor was able to internalize iodinated neurotensin. The internalization process was followed by recycling of receptors to the cell membrane. By contrast, no recycling was observed with the high affinity neurotensin receptor (NTRH). The differential intracellular routing of NTRH and NTRL after internalization is most probably the consequence of their divergent carboxy-terminal sequences.

Amino Acid Sequence↗
PubMed1982

Photoaffinity labeling of the hormone binding site of neurophysin.

The chemical structure of the hormone binding region of the neurophysins has been investigated by photoaffinity labeling with the photolabile tripeptide, L-[methyl-3H]Met-L-Tyr-p-azido-L-Phe amide. Photolysis of the photoaffinity tripeptide in the presence of bovine neurophysin I and II and a human neurophysin II led to approximately equal extents of covalent incorporation of radioactivity into protein. Photolabeled bovine neurophysin II was fractionated into binding site derivatized protein and nonbinding site derivatized protein by affinity chromatography, with results of amino acid and radiolabel analysis of the hormone binding site blocked protein indicating that 1 mol of tripeptide was covalently incorporated/mol of protein. Tyrosine 49 was the only protein amino acid modified in the binding site photolabeling reaction as assessed by peptide mapping of the performic acid oxidized and trypsin-digested photolabeled protein using reverse phase high performance liquid chromatography. Modification of the single neurophysin tyrosine also was found by amino acid analysis of performic acid oxidized photolabeled bovine neurophysin II. The covalent bond formed in neurophysin upon photolysis was cleaved by either exhaustive acid hydrolysis or reduction-carboxymethylation without loss of the protein amino acid residues and by performic acid oxidation with loss of both protein and tripeptide tyrosine residues. These overall data indicate that tyrosine 49 is the probable site for specific covalent attachment of the photoaffinity tripeptide. Assuming that the tripeptide binding site is the high affinity hormone binding site reported for the neurophysins, this conclusion argues that tyrosine 49 is close to or within this site.

Affinity Labels↗
PubMed1998

Photoaffinity labeling of cytochrome P4501A1 with azidocumene: identification of cumene hydroperoxide binding region.

Cumene hydroperoxide can support cytochrome P450-catalyzed reactions in the absence of molecular oxygen, NADPH, and cytochrome P450-NADPH oxidoreductase. Its binding at the cytochrome P450 active site is governed by the structure of the cumene hydroperoxide binding region. In order to define the region of cytochrome P4501A1 at which cumene hydroperoxide binds, we prepared an analog of cumene hydroperoxide for use as a photoaffinity label. p-Azido-isopro-pylbenzene (azidocumene) and its tritiated derivative were photolyzed in water solution by uv light with a half-life of 29 s. The 7-ethoxycoumarin deethylatation catalyzed by P450 using the cumene hydroperoxide-supported system was strongly inhibited by the presence of the label. Covalent binding to the protein after photoactivation was blocked by 50% in the presence of cumene hydroperoxide. HPLC analysis after trypsin digestion of the labeled protein showed that [3H]-azidocumene was attached covalently to the peptide VDMTPAYGLTLK corresponding to residues 492-503 in the 1A1 sequence. The radioactivity level of this fraction was reduced by 50% when the labeling was carried out in the presence of cumene hydroperoxide. To confirm the identified region the labeled protein was cleaved by cyanogen bromide. HPLC separation of the CNBr digest showed two peaks with a high level of radioactivity. The SDS/Tricine PAGE analysis of the radioactive fraction with an elution time of 43 min revealed a 2.4-kDa peptide carrying a high level of covalently bound radioactivity. The N-terminal sequence identified the labeled peptide to be a fragment generated by CNBr corresponding to residues 494-512. The N-terminal sequence of the labeled peptide with elution time of 27 min, TLKH, matches amino acid residues 501-504 in the P4501A1 sequence. We can conclude that in the overlapping region of all three identified peptides, T501-L502-K503, is the site where azidocumene covalently binds to P4501A1. The sequence alignment of cytochrome P4501A1 with cytochrome P450102 predicts that this region might correspond to beta-sheet structure localized on the distal side of the heme ring near the I helix and the oxygen binding pocket. To our knowledge, this is the first report to localize the cumene hydroperoxide binding region in the cytochrome P450 active site.

Animals↗
PubMed1990

Identification of a crotoxin-binding protein in membranes from guinea pig brain by photoaffinity labeling.

Crotoxin is a neurotoxic phospholipase A2 capable of blocking synaptic transmission by inhibiting the release of neurotransmitters. The photoaffinity labeling technique was used to identify the neural membrane molecules involved in the binding of crotoxin. A photoactivatable, radioactive derivative of crotoxin was synthesized by reacting crotoxin with N-hydroxysuccinimidyl-4-azidobenzoate and with Na[125I]. Photoirradiation of synaptosomes from guinea pig brains in the presence of the crotoxin derivative resulted in the formation of a major radioactive conjugate of 100,000 daltons as revealed by autoradiography of a sodium dodecyl sulfate-polyacrylamide gel electrophoretic pattern. Pretreatment of the synaptosomes with trypsin, Staphylococcus aureus protease, or papain prevented the formation of this conjugate. The conjugate was not detected when plasma membranes from several nonneural tissues replaced the brain synaptosomes. Unmodified crotoxin inhibited the formation of this adduct with an IC50 of about 10(-8)M. Mojave toxin, caudoxin, notexin, Naja naja PLA, and taipoxin also inhibited adduct formation with different potencies, while beta-bungarotoxin and pancreatic PLA were ineffective. We concluded that an 85,000-dalton protein is the major component responsible for the binding of crotoxin to synaptosomal membranes.

Affinity Labels↗
PubMed1980

Photoaffinity labeling of the adenosine cyclic 3',5'-monophosphate receptor protein of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate.

Photoaffinity labeling of the cAMP receptor protein (CRP) of Escherichia coli with 8-azidoadenosine 3',5'-monophosphate (8-N3cAMP) has been demonstrated. 8-N3cAMP is able to support the binding of (3H)d(I-C)n by CRP, indicating that it is a functional cAMP analogue. Following irradiation at 254 nm, (32P)-8-N3cAMP is photocross-linked to CRP. Photolabeling of CRP by (32P)-8-N3cAMP is inhibited by cAMP but not by 5'AMP. The data indicate that (32P)-8-N3cAMP is covalently incorporated following binding at the cAMP binding site of CRP. The (32P)-8-N3cAMP-CRP digested with chymotrypsin was analyzed by NaDodSO4-polyacrylamide gel electrophoresis. Of the incorporated label, one-third remains associated with the amino-proximal alpha core region of CRP [Eilen, E., Pampeno, C., & Krakow, J.S. (1978) Biochemistry 17, 2469] which contains the cAMP binding domain; the remaining two-thirds of the label associated with the beta region are digested. Limited proteolysis of the (32P)-8-N3cAMP-CRP by chymotrypsin in the presence of NaDodSO4 shows the radioactivity to be distributed between the molecular weight 9500 (amino-proximal) and 13,000 (carboxyl-proximal) fragments produced. These results suggest that a part of the carboxyl-proximal region is folded over and close enough to the cAMP binding site to be cross-linked by the photoactivated (32P)-8-N3cAMP bound at the cAMP binding site.

Affinity Labels↗
PubMed1978

Photoaffinity labeling of the angiotensin II receptor. 1. Synthesis and biological activities of the labeling peptides.

The synthesis and biological activities of analogues of the peptide hormone angiotensin II (AT) for use in photoaffinity labeling and receptor isolation are described. In the modified sequence of AT, Sar-Arg-Val-Tyr-Val-His-Pro-Phe, the aromatic residues Tyr and Phe have been either singly or simultaneously replaced by L-4'-nitrophenylalanine, L-4'-amino-3',5'-diiodophenylalanine, L-4'-aminophenylalanine, L-4'-diazoniumphenylalanine, and L-4'-azidophenylalanine. The peptides were assembled by solid-phase synthesis and the functional groups in position 4 and/or 8 chemically modified. Radioactivity was introduced by catalytic tritiation of the iodinated peptides to form the photolabeling precursors containing L-4'-amino-3',5'-diiodophenylalanine. On rabbit aorta the AT analogues substituted in position 4 showed poor affinities (0--15%), in position 8 high relative affinities (16--118%), and in position 4 and 8 additive effects of simultaneous substitutions. It is also shown that the new Boc derivative of L-4'-amino-3',5'-diiodophenylalanine can be used in peptide synthesis without side-chain protection.

Affinity Labels↗
PubMed1987

Identification of mammalian brain and spinal cord opioid receptor by photoaffinity labeling.

A radioiodinated photoreactive enkephalin derivative, 125I(D-Ala2, p-N3-Phe4-Met5)enkephalin, was used to photoaffinity label the opioid receptor from the membranes of four mammalian brains (without cerebellum) and spinal cords. These included the cat, rabbit, guinea pig and mouse. The photolabeled membranes were analyzed by sodium dodecyl sulfate gel electrophoresis. A 43,000-daltons protein was specifically photolabeled in all the membranes tested, as the specific labeling of this protein was inhibited in the presence of 14.5 microM of (D-Ala2, Met5)enkephalin. These data suggest that the 43,000-daltons protein is a binding protein of the opioid receptor in the different mammalian neural tissues.

Affinity Labels↗
PubMed1983

Response of soluble and membrane-bound F1 ATPase of Rhodospirillum rubrum to the photoaffinity label 8-azido ATP.

Soluble ATPase (F1 ATPase) and membrane-bound ATPase (chromatophores) of Rhodospirillum rubrum have been photoaffinity labeled with 8-azidoadenosine 5'-triphosphate (8-N3ATP). The specific binding of 8-N3ATP to nucleotide binding sites of the chromatophore-bound enzyme is established by competition experiments and it is suggested that hydrolysis and phosphorylation involve the same nucleotide binding sites. The experimental results also indicate that the function of the coupling factor F1 is influenced by its environment.

Adenosine Triphosphate↗
PubMed1999

Melphalan resistance and photoaffinity labelling of P-glycoprotein in multidrug-resistant Chinese hamster ovary cells: reversal of resistance by cyclosporin A and hyperthermia.

The multidrug resistance phenotype is often associated with overexpression of P-glycoprotein, an energy-dependent efflux pump responsible for decreased intracellular accumulation of chemotherapeutic agents. The role of P-glycoprotein in the mechanism of cross-resistance to melphalan in multidrug-resistant Chinese hamster ovary cells (CH(R)C5) was investigated by photoaffinity labelling of P-glycoprotein using [3H]azidopine. We investigated whether the chemosensitiser cyclosporin A and hyperthermia, either used alone or combined, could reverse melphalan resistance and alter transport processes for [14C]melphalan in CH(R)C5 cells. Melphalan inhibited azidopine photolabelling of P-glycoprotein, implicating drug efflux mediated by P-glycoprotein in the mechanism of melphalan resistance in CH(R)C5 cells. Azidopine photolabelling also was inhibited by the chemosensitiser cyclosporin A, which binds to P-glycoprotein. Cyclosporin A alone reversed melphalan resistance in CH(R)C5 cells, but had no effect in drug-sensitive AuxB1 cells. Hyperthermia (40-45 degrees) alone increased melphalan cytotoxicity in both cell lines. When hyperthermia was combined with cyclosporin A, a large increase in melphalan cytotoxicity occurred, but only in CH(R)C5 cells. This effect increased with temperature and exposure time. Sensitisation to melphalan cytotoxicity by heat and cyclosporin A in CH(R)C5 cells appeared to be explained by altered drug transport processes. Lower accumulation of melphalan occurred in CH(R)C5 cells than in drug-sensitive cells. At 37 degrees, cyclosporin A increased drug accumulation in CH(R)C5 cells, but not in AuxB1 cells, by slowing drug efflux from cells. Heat alone increased both melphalan uptake and drug efflux for both cell lines. Our findings suggest that the combination of cyclosporin A and hyperthermia could be very useful in overcoming melphalan resistance by increasing intracellular drug accumulation in multidrug-resistant cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Photoaffinity labeling of the cerebral sulfonylurea receptor using a novel radioiodinated azidoglibenclamide analogue.

In previous studies evidence has been presented by photoaffinity labeling that a polypeptide of 145-150 kDa represents the cerebral sulfonylurea receptor. However, covalent incorporation of [3H]glibenclamide or a 125I-labeled glibenclamide analogue into the sulfonylurea receptor required high amounts of photoenergy and took place with low yield of photoinsertion. To provide a probe with increased photoreactivity a 4-azido-5-iodosalicyloyl analogue of glibenclamide was synthesized. Binding experiments revealed specific and reversible high-affinity binding of this novel probe to the particulate (KD = 0.13 nM) and solubilized (KD = 0.56 nM) sulfonylurea receptor from cerebral cortex. The novel probe showed > 100-fold higher sensitivity to irradiation at 356 nm than glibenclamide. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed specific photoincorporation into a cerebral protein of 175 kDa and indicated an efficiency of photoincorporation of 9%. From dissociation binding curves following irradiation photoincorporation was estimated as 28% of specifically bound ligand. Photoincorporation into the 175-kDa protein following saturation binding of the novel probe to particulate sites from cerebral cortex indicated a KD value of 0.38 nM. Inhibition of photoincorporation into this protein by glibenclamide, glipizide, and tolbutamide revealed KD values for these sulfonylureas of 0.06 nM, 1.6 nM, and 1.2 microM, respectively. These results show that the novel photoaffinity ligand can be used as a probe for detection and characterization of the sulfonylurea receptor and suggest that a 175-kDa protein represents the cerebral sulfonylurea receptor.

ATP-Binding Cassette Transporters↗
PubMed2006

Use of photoaffinity labeling to understand the molecular basis of ligand binding to the secretin receptor.

The secretin receptor was the first member of the Class B family of G protein-coupled receptors that was identified in 1991, 89 years after secretin action was first recognized. That report resulted in the introduction of the term hormone and in the birth of the field of endocrinology. The secretin receptor has become prototypic of this receptor family, binding a moderately long linear peptide with a diffuse pharmacophoric domain. Here, we provide a detailed account of the contributions of photoaffinity labeling to establish the molecular basis of natural ligand binding to this receptor, as well as to provide insights into possible mechanisms for receptor activation and initiation of signaling. Each of the themes discussed are also relevant to other members of this physiologically and pharmacologically important receptor family.

Animals↗
PubMed1990

Purification and photoaffinity labeling of sucrose phosphate synthase from spinach leaves.

Sucrose phosphate synthase (SPS) was isolated from spinach leaves by precipitation with polyethylene glycol, ion-exchange and hydrophobic interaction chromatography, and rate zonal centrifugation. The enzyme was purified more than 600-fold to a specific activity of 57 mumol/min/mg protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a 120-kDa polypeptide was enriched through purification and was the major polypeptide in the final SPS preparation. The 120-kDa polypeptide was photoaffinity labeled with the substrate analog, 5-azidouridine [beta-32P]5'-diphosphate-glucose ([beta-32P]5-N3UDP-Glc). Covalent incorporation of 5-N3UDP-Glc into the 120-kDa polypeptide exhibited an apparent Kd of 74 microM, similar to the apparent Ki for inhibition of SPS activity by unphotolyzed 5-N3UDP-Glc. Competition experiments showed that photolabeling of the 120-kDa polypeptide by 5-N3UDP-Glc was reduced in the presence of UDP-Glc, exhibiting an apparent Ki value that was similar to the apparent Km (UDP-Glc) of 2.9 mM for the purified enzyme. The relative molecular mass of the SPS holoenzyme was 253,000, and the isoelectric point of the 120-kDa subunit was 5.2. The data confirmed the identity of the 120-kDa polypeptide as the SPS subunit, established the structure of the active enzyme as a dimer, and demonstrated active-site labeling of SPS by a photoaffinity analog of the substrate.

Affinity Labels↗
PubMed1989

First use of a beta-carboline as photoaffinity label for the benzodiazepine receptor.

Photolabelling of benzodiazepine receptors isolated from rat cortex with a new beta-carboline-type photoaffinity label, ethyl 6-azido-beta-carboline-3-carboxylase, at 254 nm produced a 42% decrease in the maximal number of propyl beta-carboline-3-carboxylate binding sites but practically no decrease in the number of flunitrazepam binding sites. Moreover, the binding affinity of ethyl beta-carboline-3-carboxylase was diminished 11-fold by photolabelling while that of diazepam was diminished less than 2-fold. These results provide additional evidence that beta-carbolines and benzodiazepines bind to discrete sites on the benzodiazepine receptor.

Affinity Labels↗
PubMed1982

Photoaffinity labeling of membrane-bound porcine aminopeptidase N.

To investigate the possible role of aminopeptidase N (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) in the transport of amino acids from oligopeptides, the modified amino acids Phe(N3) and Phe(N3, I) and the tetrapeptides Phe(N3) or Phe(N3, I)-L-or-DAla-Gly-Gly have been synthesized. The azido-amino acids were radioactively labeled by tritium or 125I before their coupling with the tripeptides. Their utilization as photoaffinity labels for aminopeptidase N has been studied. The modification imposed at the N-terminal residue of the tetrapeptides has not impaired their hydrolysis by porcine aminopeptidase N (same kinetic parameters as unmodified peptides). In addition, evidence is presented for a specific and reversible interaction in the dark of the azido-derivatives at the substrate recognition site of the enzyme. Upon photolysis, irreversible inactivation of aminopeptidase N and covalent attachment of Phe(N3, I) have been demonstrated. Soluble and membrane-bound aminopeptidases are both labeled to the same extent indicating that the free azido-amino acid preferentially reacts with the external part of the enzyme. Although the linkage of the azido-derivative is not strictly restricted to the region of the active site, the values obtained strongly suggest that 1 mol probe has been covalently attached per mol monomer of inhibited aminopeptidase.

Affinity Labels↗
PubMed1993

Photoaffinity labeling of skeletal myosin with 2-azidoadenosine triphosphate.

The purine binding site of ATP on skeletal muscle myosin has been photoaffinity labeled with 2-azidoadenosine diphosphate (2-N3ADP). 2-N3ADP was stably trapped at the active site (t1/2 approximately 5 days, 0 degree C) by complexation of the two heavy chain reactive thiols (Cys-697 and Cys-707) with Co(III)phenanthroline. Photoincorporation occurred only in the 23-kDa NH2-terminal tryptic fragment of the heavy chain. Extensive serial digestion of photolabeled subfragment 1 of myosin by trypsin and subtilisin yielded a series of labeled peptides which were purified by HPLC. Sequence and radiolabeling analysis of eight photolabeled peptides all indicated that tryptophan-130 was the only labeled residue. This site of labeling confirms earlier photolabeling studies with the non-nucleotide ADP analogue, 2[(4-azido-2-nitrophenyl)-amino]ethyl diphosphate (NANDP), which also labeled Trp-130 [Okamoto, Y., & Yount, R. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1575-1579]. Comparison of the structures of 2-N3ADP and NANDP indicate that their azido groups can be superimposed if both analogues bind to the active site in an extended conformation in a manner analogous to the anti conformation of ATP.

Adenosine Triphosphate↗
PubMed1995

Identification of the electrophilic substrate-binding site of glutathione S-transferase P by photoaffinity labeling.

We determined the electrophilic substrate-binding site of rat glutathione S-transferase P (GST-P) by photoaffinity labeling using the photosensitive compound S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione. This photosensitive glutathione analogue inhibited the catalytic activity in a competitive manner against both glutathione and 1-chloro-2,4-dinitrobenzene, a putative electrophilic substrate. The enzyme kinetics indicated that the photoactivatable glutathione analogue was specifically bound at the active site, which consisted of glutathione-binding (G-site) and the electrophilic substrate-binding (H-site) regions. The procedure involved the following steps: S-[2-(2-fluoro-4-nitrophenoxy)ethyl]glutathione was photochemically reacted with a purified recombinant GST-P expressed in Escherichia coli using ultraviolet irradiation for 30 min on ice. After the reaction, only the GST-P complexed with the glutathione analogue was prepared with glutathione-immobilized agarose. The GST-P covalently bound with the analogue was digested with lysyl endopeptidase (Achromobacter protease I), and the peptides were separated by high-performance liquid chromatography. Only a single major peak with appreciable absorbance at 340 nm was observed by peptide mapping. The peptide was collected and analyzed using an automated peptide sequencer (ABI 477A). Amino acid sequence analysis showed that this peptide consisted of seven amino acid residues corresponding to the sequence at positions 122-128 of GST-P (Ala-Leu-Pro-Gly-Xaa-Leu-Lys). No appreciable phenylthiohydantoin-amino acid was detected at the fifth cycle, which indicated that His126 was chemically labeled with the photosensitive glutathione analogue. It was concluded that His126 was one of the amino acid residues forming the electrophilic substrate-binding site of GST-P.

Amino Acid Sequence↗
PubMed2006

Characterization of the Munc13-calmodulin interaction by photoaffinity labeling.

Sensing of and response to transient increases in the residual presynaptic Ca2+ levels are important adaptive mechanisms that define the short-term plasticity characteristics of neurons. Due to their essential function in synaptic vesicle priming and in the modulation of synaptic strength, Munc13 proteins have emerged as key regulators of these adaptive mechanisms. Indeed, Munc13-1 and ubMunc13-2 contain a conserved calmodulin (CaM) binding site and the Ca2+ -dependent interaction of these Munc13 isoforms with CaM constitutes a molecular mechanism that transduces residual Ca2+ signaling to the synaptic exocytotic machinery. Here, we used Munc13-derived model peptides in photoaffinity labeling (PAL) experiments to demonstrate the stoichiometric and Ca2+ -dependent CaM binding of the other members of the Munc13 family, bMunc13-2 and Munc13-3, via structurally distinct non-conserved binding sites. A PAL-based Ca2+ titration assay revealed that all Munc13 isoforms can form a complex with CaM already at low Ca2+ concentrations just above resting levels, underscoring the Ca2+ sensor/effector function of this interaction in short-term synaptic plasticity phenomena.

Amino Acid Sequence↗
PubMed2004

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.

Amino Acid Sequence↗