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

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 Gs-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.

Glicentin-related pancreatic polypeptide inhibits glucose-stimulated insulin secretion from the isolated pancreas of adult male rats.

Peptides derived from the glucagon gene Gcg, for example, glucagon and glucagon-like peptide 1 (GLP-1), act as physiological regulators of fuel metabolism and are thus of major interest in the pathogenesis of diseases, such as type-2 diabetes and obesity, and their therapeutic management. Glicentin-related pancreatic polypeptide (GRPP) is a further, 30 amino acid Gcg-derived peptide identified in human, mouse, rat, and pig. However, the potential glucoregulatory function of this peptide is largely unknown. Here, we synthesized rat GRPP (rGRPP) and a closely related peptide, rat GRPP-like peptide (rGRPP-LP), and investigated their actions in the liver and pancreas of adult male rats by employing isolated-perfused organ preparations. Rat GRPP and rGRPP-LP did not affect glucose output from the liver, but both elicited potent inhibition of glucose-stimulated insulin secretion (GSIS) from the rat pancreas. This action is unlikely to be mediated by glucagon or GLP-1 receptors, as rGRPP and rGRPP-LP did not stimulate cyclic adenosine monophosphate (cAMP) production from the glucagon or GLP-1 receptors, nor did they antagonize glucagon- or GLP-1-stimulated cAMP-production at either receptor. GRPP and GRPP-LP may be novel regulators of insulin secretion, acting through an as-yet undefined receptor.

Identification of a pathway by which glucose regulates ß-catenin signalling via the cAMP/protein kinase A pathway in ß-cell models.

Pancreatic ß-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the ß-catenin signalling pathway plays an important role in regulating ß-cell function, but the mechanisms regulating ß-catenin signalling in these cells is not well understood. In the present study we show that ß-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and ß-TC6-F7 ß-cell models. We found a glucose-dependent increase in levels of ß-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of ß-catenin. This was associated with an increase in phosphorylation of ß-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on ß-catenin signalling. Finally, reducing ß-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate ß-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in ß-cells.

Immunohistochemical detection of the calcitonin receptor-like receptor protein in the microvasculature of rat endothelium.

Calcitonin-gene-related peptide and adrenomedullin have similar and potent vascular effects, which appear to be mediated by the G protein-coupled calcitonin receptor-like (CRL) receptor. Using immunohistochemical and Western blot analyses, we have obtained novel evidence that CRL receptor is expressed in the rat vascular endothelium using an antibody to rat CRL receptor that we have raised and fully characterised. These results are an important basis for further studies aimed at determining the so far ill-defined functional significance of the extensive distribution of CRL receptor in the vascular endothelium.