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 pbenzoyl-L-phenylalanine into the position of His 1 of rat secretin ([Bpa 1 ,Tyr 10 ]secretin-27). Because His 1 is critical for function, we also positioned a photolabile Bpa as an amino-terminal extension, in positions ؊1 (rat [Bpa ؊1 ,Tyr 10 ]secretin-27) and ؊2 (rat [Bpa ؊2 ,Gly ؊1 ,-Tyr 10 ]secretin-27). Each analog was shown to be a full agonist, stimulating cAMP accumulation in receptorbearing 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 (1-5) and to be critical for ligand binding. These disulfide bonds could provide key constraints for building a model of the secretin receptor, but definitive mapping of these bonds in an active receptor has not...

Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor

“…As such, it could function as a tethering ligand that could exert tension and thereby change the conformation of the body of the receptor that could be transmitted to the cytosolic face of the receptor where the G protein interaction occurs (59). A similar theme has been proposed for parathyroid hormone (60) and secretin (22) receptors, two other members of the same family of G protein-coupled receptors. Most recently, we have shown that the first four or five secretin amino-terminal residues were positioned close to the third extracellular loop of the receptor in our secretin-bound receptor model, with His 1 of secretin positioned at the top of transmembrane helix six (38).…”

“…receptors for follicle-stimulating hormone (56,57), luteinizing hormone/choriogonadotropin (52), and gonadotropin-releasing hormone (58)) to activate these receptors. It is noteworthy that the third extracellular loop of G protein-coupled receptors is near the sixth transmembrane domain, which has been shown to interact directly with the amino terminus of the peptide ligand for receptors for parathyroid hormone (23,24) and secretin (22).…”

“…Active Site Identification-To gain an initial indication of receptor domains of labeling with the Bpa 8 probe, we first chose to cleave the labeled receptor by CNBr that we have used successfully for the secretin (22,26,(35)(36)(37)(38)(39) and CT (20) receptors in our laboratory. CNBr cleaves at the carboxyl side of Met residues within a protein, and its cleavage of the CT receptor would theoretically yield 15 fragments ranging from 0.1 to 11 kDa, two of which contain potential glycosylation sites.…”

“…This new pair of residue-residue approximation should contribute substantially to our understanding of the natural ligand-binding region of the CT receptor. Analogous observations with the structurally related secretin (22) and parathyroid hormone (23,24) receptors may suggest a common mechanism for ligand binding and activation of the class B family of G protein-coupled receptors.…”

Molecular Basis of Glucagon-like Peptide 1 Docking to Its Intact Receptor Studied with Carboxyl-terminal Photolabile Probes

“…Based on these studies, a "two-step" or "tethering" mechanism has been proposed, where the carboxyl-terminal region of the peptide ligand initially interacts with the amino-terminal domain of the receptor, and in the second step, the amino-terminal region of the ligand interacts with the core domain of the receptor, which leads to receptor activation (27). This mechanism has also been proposed for other members of Family B GPCRs (42)(43)(44). Our current data show that both the position 24 and 35 residues of GLP1 are in proximity with the residues in the amino-terminal domain of its receptor, which is consistent with this mechanism.…”