Multiple Sites of Contact between the Carboxyl-terminal Binding Domain of PTHrP-(1–36) Analogs and the Amino-terminal Extracellular Domain of the PTH/PTHrP Receptor Identified by Photoaffinity Cross-linking

Abstract

The carboxyl-terminal portions of parathyroid hormone (PTH)-(1-34) and PTH-related peptide (PTHrP)-(1-36) are critical for high affinity binding to the PTH/ PTHrP receptor (P1R), but the mechanism of receptor interaction for this domain is largely unknown. To identify interaction sites between the carboxyl-terminal region of PTHrP-(1-36) and the P1R, we prepared analogs of [ -172. These data thus predict that residues 23, 27, 28, and 33 of native PTHrP are each near to different regions of the amino-terminal extracellular receptor domain of the P1R. This information helps define sites of proximity between several ligand residues and this large receptor domain, which so far has been largely excluded from models of the hormone-receptor complex. PTH and PTHrP1 mediate many of their biological effects through the same receptor (1, 2). Peptides containing the first 34 amino acids of PTH and PTHrP are capable of fully activating the PTH/PTHrP receptor (P1R) (1, 2). Studies with PTH and PTHrP ligand analogs and receptor chimeras have suggested that the ligands have two distinct functional domains: the amino-terminal residues, which are important for receptor activation; and the carboxyl-terminal residues, which are important for high affinity binding (3). These data furthermore indicate that the ligand's amino-terminal portion interacts with the extracellular loops and the membrane-spanning helices of the receptor, and the ligand's carboxyl-terminal portion interacts with the receptor's amino-terminal extracellular domain (3). A similar pattern of ligand-receptor interaction has been suggested for other members of this class II family of peptide hormone G-protein-coupled receptors, including the secretin receptor (4) and the PTH-2 receptor (5-7).Some specific sites of interaction between the amino-terminal portions of PTH-(1-34)/PTHrP-(1-36) and the P1R have been identified by site-directed mutagenesis and photoaffinity cross-linking studies. For example, mutational analyses have shown that residues in extracellular loop 3 and the adjacent sixth membrane-spanning helix (TM6) are critical for mediating ligand-induced receptor activation (2,8). Consistent with these mutational data, Bpa introduced at position 1 of PTH-(1-34) or position 2 of either PTH-(1-34) or PTHrP-(1-36) was found to cross-link to methionine 425, at the extracellular end of TM6 in the P1R (9, 10). Interactions between the aminoterminal portion of the ligand and the transmembrane domains/extracellular loops of the receptor are also suggested by a study showing that PTH-(1-14) can stimulate cAMP accumulation in a mutant P1R missing most of the amino-terminal extracellular domain as efficiently as it does in the wild-type P1R (11). Furthermore, if a PTH fragment comprising the first 9 amino acids is covalently attached to the amino-terminal end of such a truncated receptor, the resulting ligand-receptor chimera displays constitutive activity, indicative of an intramolecular stimulation of the receptor's activation domain by the tethered ligand fragment ...

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

“…The carboxyl-terminal position 33 PTH probe has been demonstrated to label the receptor amino terminus adjacent to the first transmembrane domain (49), consistent with labeling the GLP1 receptor with the carboxyl-terminal probes in this study. However, another two carboxyl-terminal position 23 and 28 probes were shown to label the distal amino terminus (49,50). The labeling of the calcitonin receptor is quite consistent with the data from the secretin and PTH receptors (44,51), but the labeling of the VPAC1 receptor suggests a different mechanism.…”

“…Like the secretin receptor, while the midregion and carboxyl-terminal PTH or PTH-related peptide probes have been shown to label residues within the amino terminus of the PTH receptor, the amino-terminal probe has been shown to label residues within the sixth transmembrane domain (42,47,48). The carboxyl-terminal position 33 PTH probe has been demonstrated to label the receptor amino terminus adjacent to the first transmembrane domain (49), consistent with labeling the GLP1 receptor with the carboxyl-terminal probes in this study. However, another two carboxyl-terminal position 23 and 28 probes were shown to label the distal amino terminus (49,50).…”

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 hypothesis that the VIP-binding cleft could reside between the hVPAC1 receptor N-terminal ectodomain for the central part of VIP (this paper and Ref. 8) and the receptor body for the N-terminal domain of VIP is reminiscent of the situation described for some other class II peptide receptors (22)(23)(24)(25).…”

Structure of the N-terminal domain of a type B1 G protein-coupled receptor in complex with a peptide ligand

“…The modular property is also present for various peptide hormones such as parathyroid hormone (45). Typically the N-terminal segment of the ligand is responsible for receptor signaling, whereas the C-terminal segment is an important determinant for the receptor binding (45,46) and may interact with the ECD1 of the receptor as evidenced by cross-linking (47). Furthermore, the bioactive conformations of various peptide hormones are proposed to be of amphipathic helical nature (48), similar to the conformation observed for astressin.…”