Cryo-EM structure of the active, Gs-protein complexed, human CGRP receptor

Abstract

Calcitonin gene-related peptide (CGRP) is a widely expressed neuropeptide that has a major role in sensory neurotransmission. The CGRP receptor is a heterodimer of the calcitonin receptor-like receptor (CLR) class B G-protein-coupled receptor and a type 1 transmembrane domain protein, receptor activity-modifying protein 1 (RAMP1). Here we report the structure of the human CGRP receptor in complex with CGRP and the G-protein heterotrimer at 3.3 Å global resolution, determined by Volta phase-plate cryo-electron microscopy. The receptor activity-modifying protein transmembrane domain sits at the interface between transmembrane domains 3, 4 and 5 of CLR, and stabilizes CLR extracellular loop 2. RAMP1 makes only limited direct contact with CGRP, consistent with its function in allosteric modulation of CLR. Molecular dynamics simulations indicate that RAMP1 provides stability to the receptor complex, particularly in the positioning of the extracellular domain of CLR. This work provides insights into the control of G-protein-coupled receptor function.

An Inverse Agonist Ligand of the PTH Receptor Partially Rescues Skeletal Defects in a Mouse Model of Jansen's Metaphyseal Chondrodysplasia

“…These structures thus show the peptide to be bound as a linear α-helix with its Cterminal portion docked to the ECD and its N-terminal portion projecting into the core of the TMD bundle. This binding mode is similar to that seen in the high-resolution structures reported for several other family B GPCRs, including the receptors for glucagon, GLP-1, and calcitonin, each in complex with a bound peptide ligand, (14)(15)(16)(17)(18) thus supporting a common basic mechanism of ligand-induced activation used by each of the family B GPCRs.…”

“…1A. In functional studies in vitro, [Leu 11 ,dTrp 12 ,Trp 23 ,Tyr 36 ]-PTHrP(7-36)NH 2 was more effective as an inverse agonist than the PTH-based analog, [Nle 8,18 ,dTrp 12 , Tyr 34 ]-bPTH(7-34)NH 2, (24,25) because it more effectively reduced basal cAMP signaling in HEK293 cells stably transfected to express PTHR1-H223R and the GloSensor cAMP reporter ( Fig. 1B,C).…”

“…PTHR1 is a family B G-protein-coupled receptor (GPCR) and, as for each of these receptors, binds its agonist peptide ligands, such as the bioactive PTH(1-34) fragment, via a two-site mechanism. This mechanism involves an initial docking of the (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34) portion of the ligand to the amino-terminal extracellular domain (ECD) portion of the receptor and a subsequent engagement of the N-terminal (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) portion of the ligand with the extracellular loop and seven-transmembrane helical domain (TMD) region of the receptor, which leads to receptor activation. (10,11) The recent high-resolution X-ray crystal (12) and cryogenic electron microscopy (cryo-EM) (13) structures of the PTHR1, each in complex with a PTH or PTHrP analog ligand, confirm and extend this model of ligand binding.…”

A potent fluorescent calcitonin gene‐related peptide analogue enables visualization of receptor internalization

“…Furthermore, the potency of [TAMRA] 3 ‐hαCGRP 3 at both receptors was approximately equal to that of hαCGRP with no significant losses in potency, indicating that the addition of a fluorophore moiety is well‐tolerated at position 3 by CGRP. In the cryo‐EM structure of the peptide‐bound CGRP receptor, the side‐chain of Asp‐3 appears to reside in an exposed area, providing a possible explanation for the potent activity of this analogue . The structure of the AMY 1 receptor is currently unknown, although substitution of the asparagine at position 3 of human amylin with alanine is without effect .…”

Adrenomedullin disulfide bond mimetics uncover structural requirements for AM1 receptor activation

“…This residue is highly conserved throughout the peptide family (Thr 6 in AMY and CGRP, Thr 14 in IMD), and its particular importance for receptor activation has been highlighted by several studies . In fact, a recently published structure of CGRP bound to the active‐state CGRPR revealed a functionally important contact between the corresponding Thr 6 of CGRP and His 295 of the CLR . A similar function of ADM Thr 20 is very likely.…”