Spatial Approximation between Secretin Residue Five and the Third Extracellular Loop of Its Receptor Provides New Insight into the Molecular Basis of Natural Agonist Binding

Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, 13400 East Shea Blvd., Scottsdale, AZ 85259, USA.
Molecular pharmacology (Impact Factor: 4.13). 06/2008; 74(2):413-22. DOI: 10.1124/mol.108.047209
Source: PubMed


The amino terminus of class II G protein-coupled receptors plays an important role in ligand binding and receptor activation. Understanding of the conformation of the amino-terminal domain of these receptors has been substantially advanced with the solution of nuclear magnetic resonance and crystal structures of this region of receptors for corticotrophin-releasing factor, pituitary adenylate cyclase-activating polypeptide, and gastric inhibitory polypeptide. However, the orientation of the amino terminus relative to the receptor core and how the receptor gets activated upon ligand binding remain unclear. In this work, we have used photoaffinity labeling to identify a critical spatial approximation between residue five of secretin and a residue within the proposed third extracellular loop of the secretin receptor. This was achieved by purification, deglycosylation, cyanogen bromide cleavage, and sequencing of labeled wild-type and mutant secretin receptors. This constraint has been used to refine our evolving molecular model of secretin docked at the intact receptor, which for the first time includes refined helical bundle and loop regions and reflects a peptide-binding groove within the receptor amino terminus that directs the amino terminus of the peptide toward the receptor body. This model is fully consistent with the endogenous agonist mechanism for class II G protein-coupled receptor activation, where ligand binding promotes the interaction of a portion of the receptor amino terminus with the receptor body to activate it.

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    • "Crystallography (Sasaki et al., 1975; Parthier et al., 2007) and nuclear magnetic resonance (NMR; Grace et al., 2007; Venneti and Hewage, 2011) analyses have also been useful, elucidating the class B ligands to preferentially assume an α-helical conformation particularly in their C-terminal regions. Structure–activity studies have further implicated the specific interaction of ligand–receptor regions, with the C-terminal ligand domain first fitting into the receptor ECD peptide-binding cleft, thus facilitating the N-terminal ligand domain to associate with the receptor core helical bundle domain (Dong et al., 2004, 2008; Mann et al., 2007; Parthier et al., 2009). Though the molecular interactions described are on the basis of monomeric GPCRs, similar associations may be expected from oligomeric forms. "
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    ABSTRACT: Dimerization or oligomerization of G protein-coupled receptors (GPCRs) are known to modulate receptor functions in terms of ontogeny, ligand-oriented regulation, pharmacological diversity, signal transduction, and internalization. Class B GPCRs are receptors to a family of hormones including secretin, growth hormone-releasing hormone, vasoactive intestinal polypeptide and parathyroid hormone, among others. The functional implications of receptor dimerization have extensively been studied in class A GPCRs, while less is known regarding its function in class B GPCRs. This article reviews receptor oligomerization in terms of the early evidence and current understanding particularly of class B GPCRs.
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    ABSTRACT: Corticotropin-releasing factor receptor 1 (CRFR1) mediates the physiological actions of corticotropin-releasing factor in the anterior pituitary gland and the central nervous system. Using chemical cross-linking we have previously reported that residue 16 of sauvagine (SVG) is in a close proximity to the second extracellular loop of CRFR1. Here we introduced p-benzoylphenylalanine (Bpa) at position 17 of a sauvagine analog, [Tyr0, Gln1, Bpa17]SVG, to covalently label CRFR1 and characterize the cross-linking site. Using a combination of receptor mutagenesis, peptide mapping, and N-terminal sequencing, we identified His117 within the first transmembrane domain (TM1) of CRFR1 as the cross-linking site for Bpa17 of 125I-[Tyr0, Gln1, Bpa17]SVG. These data indicate that, within the SVG-CRFR1 complex, residue 17 of the ligand lies withina9Å distance from residue 117 of the TM1 of CRFR1. The molecular proximity between residue 17 of the ligand and TM1 of CRFR1 described here and between residue 16 of the ligand and the CRFR1 second extracellular loop described previously provides useful molecular constraints for modeling ligand-receptor interaction in mammalian cells expressing CRFR1.
    Journal of Biological Chemistry 11/2008; 283(51):35644-51. DOI:10.1074/jbc.M806351200 · 4.57 Impact Factor
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    ABSTRACT: The efficiency of covalent labeling of a receptor by a photolabile analogue of its natural ligand is dependent on the spatial approximation of the probe and its target. Systematic application of intrinsic photoaffinity labeling to the secretin receptor, a prototypic Family B G protein-coupled receptor, demonstrated reduced efficiency of labeling for amino-terminal and mid-region sites of labeling relative to carboxyl-terminal sites. Reduction of pH from 7.4 to 5.5 and reduction of temperature from 25 degrees C to 4 degrees C improved the efficiency of covalent labeling of the receptor with these probes. This correlated with sites of labeling at the interface between the receptor amino terminus and the receptor core, a region containing histidine residues that have their ionization affected in this pH range. Application to the calcitonin receptor, another Family B G protein-coupled receptor, yielded analogous results. These results support the consistent mode of docking peptide ligands to this group of receptors.
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