Repository logo

Influence of Peptide Allosteric Modulators on Agonist Bias at Class B1 G Protein-Coupled Receptors



Change log



Class B1 G Protein-Coupled Receptors (GPCRs) are a small family within the GPCR superfamily. However, they are implicated in the pathologies of some of the most prevalent diseases, such as type 2 diabetes and heart disease. Despite being such a small family, their signalling is very diverse; each receptor responds to multiple endogenous agonists and couples to different G proteins, displaying pleiotropy. There is added variation in receptor desensitisation and internalisation, with intracellular signalling and β-arrestin-mediated pathways adding spatial and temporal complexity. How this crosstalk regulates intracellular signalling was investigated at the Glucagon-Like Peptide-1 Receptor (GLP1R), a Class B1 GPCR with implications in glucose homeostasis. Its G protein-dependent signalling was measured, activating a wide range of G proteins, not confined to a certain subfamily. β-arrestin recruitment and internalisation were examined, with GLP1R undergoing rapid internalisation, with a complicated dependency on β-arrestins. Insulin secretion was also measured, and the role of receptor desensitisation examined in this downstream response. Reducing internalisation correlated with a reduction in insulin secretion.

Genetic variation in Class B1 GPCRs can lead to differences in signalling. Single Nucleotide Polymorphisms (SNPs) resulting in missense mutations can directly alter agonist or G protein binding, or alter stabilisation of active and inactive receptor conformations through allosteric mechanisms. The consequences of SNPs in N-terminal and C-terminal regions of GLP1R, glucagon receptor (GCGR), secretin receptor (SCTR), and corticotropin-releasing factor receptor type 1 (CRF1) were therefore investigated. Whilst some effects were observed when GLP1R was mutated, in many cases these SNPs had little effect on signalling. However, mutation of a conserved residue, arginine3.30, was severely detrimental to GLP1R signalling. In addition to SNPs, large genetic variation is found in the form of splice isoforms. A GCGR splice isoform found in human cells was shown to express poorly, displaying little signalling. However, its co-expression altered signalling of the reference GCGR, reducing G protein signalling but increasing β-arrestin recruitment, showing how dimer formation alters agonist binding.

In addition to internal variation, the expression of peptide modulators such as receptor activity-modifying proteins (RAMPs) can greatly influence Class B1 GPCR pharmacology. The effect of RAMP3 expression on GLP1R signalling and desensitisation was measured, with increases in Gαq coupling and intracellular Ca2+ (Ca2+)i mobilisation mediated by GLP1R transiently expressed in HEK293T cells. (Ca2+)I mobilisation was also increased by RAMP3 overexpression in INS-1 832/3 cells, which endogenously express the receptor. Increased Gαq/11 signalling increased insulin secretion in response to GLP1. The calcitonin-like receptor (CLR) is known to interact with all three RAMPs to generate functionally distinct receptors. Biased G protein-mediated signalling of CLR has been well-studied, but the role of RAMPs in CLR desensitisation and internalisation has been relatively unexamined. A global characterisation of CLR-RAMP internalisation in response to the three primary endogenous agonists was therefore achieved. The mechanism of internalisation was elucidated, and its role in cAMP signalling tested. GPCR-kinases (GRKs) are important in GPCR β-arrestin recruitment and subsequent internalisation. Attempts to identify which GRKs are responsible for phosphorylation of CLR instead identified constitutive phosphorylation and internalisation of the receptor.

This study includes several different allosteric means to regulate Class B1 GPCR signalling. Mutation of residues outside the orthosteric binding site can change G protein coupling, even without interference of the interacting residues. However, more common peptide allosteric modulators are those co-expressed with the receptor, such as RAMPs and GRKs.





Ladds, Graham


Agonist Bias, Diabetes Mellitus, G protein-Coupled Receptor, Insulin


Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Biotechnology and Biological Sciences Research Council (2120024)
Biotechnology and Biological Sciences Research Council (2100523)
BBSRC (2120024)