Structure and mechanism for recognition of peptide hormones by Class B G-protein-coupled receptors

Abstract

Class B G-protein-coupled receptors (GPCRs) are receptors for peptide hormones that include glucagon, parathyroid hormone, and calcitonin. These receptors are involved in a wide spectrum of physiological activities, from metabolic regulation and stress control to development and maintenance of the skeletal system. As such, they are important drug targets for the treatment of diabetes, osteoporosis, and stress related disorders. Class B GPCRs are organized into two modular domains: an extracellular domain (ECD) and a helical bundle that contains seven transmembrane helices (TM domain). The ECD is responsible for the high affinity and specificity of hormone binding, and the TM domain is required for receptor activation and signal coupling to downstream G-proteins. Although the structure of the full-length receptor remains unknown, the ECD structures have been well characterized for a number of Class B GPCRs, revealing a common fold for ligand recognition. This review summarizes the general structural principles that guide hormone binding by Class B ECDs and their implications in the design of peptide hormone analogs for therapeutic purposes.

Use of Cysteine Trapping to Map Spatial Approximations between Residues Contributing to the Helix N-capping Motif of Secretin and Distinct Residues within Each of the Extracellular Loops of Its Receptor

“…The first two crystal structures of isolated helical bundles of the glucagon and corticotropin releasing factor-1 receptors were finally reported in 2013, confirming differences that had been predicted from the helical bundles of class A GPCRs (18,33). It is important to note that the orientation of the receptor amino-terminal domain relative to its core transmembrane domain of the class B GPCRs is still poorly understood, as demonstrated by a highly diverse set of proposed holoreceptor structures (5,30). Adding to this uncertainty is the prediction that the helical bundle of the class B G protein-coupled receptors will be quite different from that of the class A receptors, now well defined in the crystal structures (18,33).…”

“…Our best insight into the class B structure came from the crystallization of the extracellular amino-terminal domains of several members of the B1 group (30,31), some of these also including complexed peptide ligands (30,31). Indeed, these observations have confirmed the presence of a highly conserved conformational motif, called a "sushi motif," which is formed by three intradomain disulfide bonds linking two antiparallel ␤ sheet regions and various loops as well as a less consistent amino-terminal helix (30,31). This structure provides a hydrophobic cleft for the docking of the carboxylterminal region of the natural peptide ligands in ␣-helical conformation (32).…”

Differential Requirement of the Extracellular Domain in Activation of Class B G Protein-coupled Receptors

“…The encoded fragments were digested with BamHI and NotI restriction endonucleases and inserted into a modified pcDNA6 expression vector to encode a fusion protein consisting of an N-terminal human IgG leader (MGWSCIILFL-VATATGVHSE) for targeting into the cell membrane and a FLAG tag (DYKDDDD) at the C terminus for detection by immunoblotting. In addition to the full-length receptors, we generated the same set of constructs with (i) the TMDs of GCGR (residues 123-431), GLP-1R (residues 140 -463), PTH1R (residues 182-484), CRF 1 R (residues 110 -384), and PAC1R (residues 148 -421), (ii) fusions between the N-terminal peptide hormones (glucagon (1-15), GLP-1(7-21), PTH(1-15), UNC (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), PACAP (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)) and their corresponding TMDs, (iii) fusions of the same N-terminal peptide fragments as above to the BRIL-TMD constructs, and (iv) fusions of the full-length peptide hormones to the N terminus of their corresponding full-length receptors with five copies of Gly-Ser-Ala (GSA 5 ) linker. All these constructs contain the same IgG leader as above.…”

“…Signaling-To examine the requirement of the GCGR ECD for signaling, we transiently transfected four different FLAGtagged constructs into HEK293 cells: the full-length receptor, TMD, TMD-interacting N terminus of glucagon (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) fused to the TMD, and a fusion in which glucagon (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15) and the TMD are separated by a thermostabilized cytochrome b562 variant, BRIL, which has been used for facilitating GPCR crystallization (16) (Fig. 1, B-D).…”

“…Structures of the ECDs of class B GPCRs in complex with their peptide ligands have been determined by x-ray crystallography (1, 6 -9) and NMR (10) and have provided useful information about structural mechanisms of ligand recognition and selectivity (2,11). Only recently, crystal structures of the iso-lated TMDs of two class B receptors in an inactive conformation have been published, i.e.…”