Spatial Approximation between the Amino Terminus of a Peptide Agonist and the Top of the Sixth Transmembrane Segment of the Secretin Receptor

Abstract

Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-l-phenylalanine into the position of His1 of rat secretin ([Bpa1,Tyr10]secretin-27). Because His1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa-1,Tyr10]secretin-27) and -2 (rat [Bpa-2,Gly-1,Tyr10]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation. Distinct spatial approximations between residues within the secretin pharmacophore and its receptor can provide important constraints for modeling this agonist-receptor complex. We previously used a series of probes incorporating photolabile residues into positions 6, 12, 13, 14, 18, 22, and 26 of the 27-residue peptide and demonstrated that each covalently labeled a site within the receptor amino terminus. Although supporting a critical role of this domain for ligand binding, it does not explain the molecular mechanism of receptor activation. Here, we developed probes having photolabile residues at the amino terminus of secretin to explore possible approximations with a different receptor domain. The first probe incorporated a photolabile p-benzoyl-l-phenylalanine into the position of His1 of rat secretin ([Bpa1,Tyr10]secretin-27). Because His1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions -1 (rat [Bpa-1,Tyr10]secretin-27) and -2 (rat [Bpa-2,Gly-1,Tyr10]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptor-bearing Chinese hamster ovary-SecR cells in a concentration-dependent manner, with the position -2 probe being most potent. They bound specifically and saturably, although the position 1 analog had lowest affinity, and all were able to label the receptor efficiently. Sequential specific cleavage, purification, and sequencing demonstrated that the sites of covalent attachment for each probe were high within the sixth transmembrane segment. This suggests that secretin binding may exert tension between the receptor amino terminus and the transmembrane domain to elicit a conformational change effecting receptor activation. The secretin receptor is prototypic of the Class B family of guanine nucleotide-binding protein (G protein)-coupled receptors which includes many important drug targets. Understanding of the molecular basis of ligand binding of receptors is important for the rational design of receptor-active drugs. However, the molecular basis of ligand binding of Class B receptors is less well understood than that of members of the Class A family, such as rhodopsin and the adrenergic receptor. This likely reflects the facts that natural ligands for Class B G protein-coupled receptors are relatively large peptides with diffuse phamacophoric domains and that these receptors have long and complex amino-terminal domains that are important for binding. Both of these interacting domains are flexible, with active conformations that have not been clearly defined. One of the distinct characteristics of the Class B receptor family is the long amino terminus that exceeds 120 residues in length. It contains 6 conserved Cys residues that have been demonstrated to form intradomain disulfide bonds (1.Qi L.J. Leung A.T. Xiong Y. Marx K.A. Abou-Samra A.B. Biochemistry. 1997; 36: 12442-12448Crossref PubMed Scopus (69) Google Scholar, 2.Perrin M.H. Fischer W.H. Kunitake K.S. Craig A.G. Koerber S.C. Cervini L.A. Rivier J.E. Groppe J.C. Greenwald J. Moller N.S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 3.Grauschopf U. Lilie H. Honold K. Wozny Biochemistry. PubMed Scopus Google Scholar, U. Wozny Chem. PubMed Scopus Google Scholar, L.J. PubMed Scopus Google and to be critical for ligand binding. disulfide bonds provide constraints for a of the secretin of these bonds in an active receptor not been A been for Class B G protein-coupled binding used rat rat rat secretin receptor-bearing Chinese hamster high transmembrane domain. U. Lilie H. Honold K. Wozny Biochemistry. PubMed Scopus Google peptide U. Wozny Chem. PubMed Scopus Google and M.H. Fischer W.H. Kunitake K.S. Craig A.G. Koerber S.C. Cervini L.A. Rivier J.E. Groppe J.C. Greenwald J. Moller N.S. Vale W.W. J. Biol. Chem. 2001; 276: 31528-31534Abstract Full Text Full Text PDF PubMed Scopus (79) Google of the amino-terminal peptides of these receptors that had been in It be that the of disulfide bonds the amino-terminal domain of the is different that of that receptor (1.Qi L.J. Leung A.T. Xiong Y. Marx K.A. Abou-Samra A.B. Biochemistry. 1997; 36: 12442-12448Crossref PubMed Scopus (69) Google that of members of the Class B family, the complex amino terminus of the secretin receptor been demonstrated to a critical role in ligand binding and receptor M.H. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.H. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google We have a to the molecular basis of secretin binding to its This can provide spatial approximations between photolabile residues within a ligand and residues to these at the receptor. We have used a series of probes with a photolabile in different of the in positions 6, 12, 13, 14, 18, 22, and 26 Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google all of these probes have been demonstrated to label residues within the amino-terminal domain of the secretin with probes the first residues of the amino terminus Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and a to the first transmembrane L.J. PubMed Scopus Google This a critical role for the amino terminus of the secretin receptor in ligand binding. However, have shown in to the amino-terminal of the secretin the that ligand binding. It is not such are interacting with the interacting with of the receptor Because the amino-terminal of secretin been shown to be important and not been in the we this as a possible site of with the of the receptor. The amino terminus of the natural ligand of Class B G protein-coupled been shown to with the of the sixth transmembrane of that receptor L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 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L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google The site of with the probe was specifically to its receptor the and labeled the receptor residues and modeling L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we have a of the secretin receptor and the secretin peptide the Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. 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PubMed Scopus Google Each of these probes labeled residues within the first residues of the amino-terminal domain of the secretin receptor. we also that probe with the photolabile incorporated into the of the in position 13, also labeled an amino-terminal in the secretin receptor L.J. PubMed Scopus Google However, this was to the first transmembrane than at the of this domain L.J. PubMed Scopus Google have the of the amino-terminal domain of this receptor at the a role for an to secretin be interacting with such of the receptor as well as the amino we to this we the amino-terminal of a not previously However, of a photolabile in the position of the first of secretin a The in this position been shown to be critical for binding and in H. 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Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (79) Google One possible was to the amino terminus of the probes to receptor binding, was that this also with the receptor. we developed a to the amino within the probe and the labeled receptor to to a receptor that be to this the photolabile amino-terminal probes bound to the receptor to the amino of the photolabile ligand and the secretin receptor were the labeled receptor to sequencing Because the probes had this we were able to the specific receptor residues covalently labeled each of the amino-terminal photolabile all probes labeled a and receptor domain. The probe labeled the and probes labeled receptor residues and Each of these residues is to high in the sixth transmembrane segment. This is the first secretin receptor domain covalently labeled in such an which is distinct the amino which had been labeled each of the previously probes Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google have an important role for the of the sixth transmembrane within the Class B G protein-coupled receptor H. PubMed Google Scholar, H. Abou-Samra PubMed Scopus Google This domain also been shown to be important for Class A such as the receptor Biochemistry. 1997; 36: PubMed Scopus Google and the receptor U. PubMed Scopus Google The of the sixth transmembrane was also labeled in a series of of the receptor. probes with a Bpa at the amino terminus of in position 1 L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google in position J. Biol. Chem. 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PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google the between the peptide terminus and the amino-terminal domain of the receptor. The the between the peptide amino terminus and the of the receptor. However, with the probe may a Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google probe also labeled the receptor amino terminus. a molecular of the complex the and between these modeling to the first complex which was the the first secretin amino-terminal residues were positioned to the receptor with His1 positioned at the of transmembrane the the mechanism that the binding of secretin ligand the domains of the the amino terminus and transmembrane and suggests that the mechanism may well be to that for the we have developed amino-terminal photolabile secretin probes and have to approximations as at the secretin receptor. Each probe covalently labeled a the of the of the secretin a that is distinct that labeled probes previously Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Y. L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. PubMed Scopus Google Scholar, L.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we have critical spatial constraints that are for the modeling of the secretin receptor. a mechanism for receptor to that for the the peptide distinct domains of the a mechanism to exert tension and change This may be a for the Class B G protein-coupled We and for