Mechanisms of GPCR Signal Transduction

Novel effects of allosteric modulators on GPCR trafficking and regulation

It is now acknowledged that the clinical effects of many therapeutic agents are not solely determined by their acute effects on receptor signaling, but also by longer term effects that they can exert on receptor regulation events, such as phosphorylation, trafficking and changes in receptor expression levels. We have recently found evidence that some allosteric modulators of the M2 muscarinic acetylcholine receptor (mAChR) can alter the cell-surface expression of the receptor after long-term drug exposure. This finding has important implications for the therapeutic use of allosteric modulators of GPCRs, because drug treatments usually result in receptors being exposed to drugs for prolonged periods of time. We are currently investigating the extent of this phenomenon and the mechanisms underlying it. We are also investigating the effects of novel allosteric agonists, that is, ligands that bind to an allosteric site on the mAChRs but activate the receptor in their own right, to determine whether the effects of such agents on receptor signalling and regulation differ and/or are more selective than those of prototypical orthosteric agonists.

Characterization of RAMP-receptor interactions

The peptides classically designated as calcitonin peptide family members include calcitonin gene-related peptide (CGRP), amylin (AMY) and adrenomedullin (AM), although an assortment of related peptides have also been identified. However, until recently, the molecular nature of the cognate receptors for AMY, CGRP and adrenomedullin was unknown. Receptors for the calcitonin family of peptides arise from heterodimerisation of the calcitonin GPCR (CTR) or the calcitonin-like GPCR (CLR) with a family of 3 accessory proteins termed receptor activity modifying proteins (RAMPs), with distinct receptor phenotypes arising from individual RAMP-receptor combinations. In addition to identifying the CTR-RAMP interaction, we have recently demonstrated that RAMPs interact with at least 4 other GPCRs and have shown that an additional action of RAMPs may be to selectively alter signal pathway activation. We are currently investigating the structural basis and functional consequences of these novel interactions in collaboration with Dr. Debbie Hay (Univ. Auckland, NZ).

Characterization of Family B GPCRs

GPCRs can be broadly subclassified into 3 major families. Family A receptors have homology to rhodopsin across the 7 transmembrane spanning domains and this group is the largest and most diverse subfamily of GPCRs including receptors for amines, small peptides, large glycoprotein hormones, fatty acids, cytokines, and proteases. The smallest GPCR family is Family C, which are receptors for small molecules such as GABA, glutamate or calcium. The second largest GPCR subfamily is the Family B group of receptors. Of these, the best studied are the peptide hormone subgroup that includes receptors for peptides such as calcitonin, secretin, parathyroid hormone, vasoactive intestinal polypeptide, glucagon and glucagon-like peptides. There is increasing evidence that GPCRs, particularly Family A and Family C receptors, exist as constitutive dimers both homomeric and heteromeric. Less is known about the functional unit of Family B receptors. In collaboration with Prof. Larry Miller’s group (Mayo Clinic, Scottsdale, Az) we have recently shown that most Family B peptide hormone receptors can form homodimers and that many can form heterodimers with other Family B receptors. The degree to which this occurs, however, is receptor specific. One example of this is the secretin receptor, which forms homodimers and heterodimers with the VPAC1 and VPAC2 receptors. Furthermore, the VPAC1 and VPAC2 receptors heterodimerise and both these heterodimers and the VPAC1 or VPAC2 homodimers can be modulated by agonist, but not antagonist, treatment. In contrast, secretin receptor based dimers are not modulated. The functional significance of these interactions is currently being explored.

Biophysical studies of GPCR-G protein coupling

Although it is well recognised that GPCRs may couple to multiple G proteins, and thus to multiple signal transduction pathways, the full spectrum of potential interactions for an individual receptor has not been well explored. Moreover, most conclusions about receptor-G protein coupling specificity need to be inferred indirectly from studies that monitor downstream signalling events. In collaboration with Prof. Michel Bouvier (University of Montreal) and Dr. Celine Gales (INSERM U388, Toulouse, France), we have developed novel methods using Bioluminescence Resonance Energy Transfer (BRET) to explicitly assess direct interactions between GPCRs and individual G proteins in real time and in living cells. This enables, for the first time, examination of the specificity of ligand responses at the most proximal level of receptor signal transduction for the entire repertoire of human G proteins, and can be used to provide direct evidence for distinct conformational states (which may underlie distinct physiological responses) being induced by individual ligands.

GPCR signaling promiscuity and trafficking of receptor stimulus

It is now recognized that most GPCRs can activate multiple intracellular second messenger pathways that may be G protein dependent or in some cases G protein independent. Traditionally, it was viewed that different classes of drugs acting at the receptor may exhibit a range of potency and efficacy from full agonist, to partial agonist, to neutral antagonist and in the case of constitutively active systems, partial and full inverse agonists. However, the spectrum of pathway activation was either assumed to be similar across pathways or else principally limited to one pathway (eg. cAMP formation). There is now accumulating evidence that this latter assumption is not correct and that specific drugs may be able to favour individual signaling pathways in a drug-specific manner, leading to what has been described as biased agonism or agonist-directed trafficking of receptor stimulus (ADTRS); this may also be extended to drug specific modulation of receptor regulation. Understanding the capacity of individual drugs to selectively activate specific signalling pathways, or differentially affect receptor regulation may provide unique opportunities for the development of more selective and/or safer pharmaceuticals. We are applying a variety of approaches to understand the capacity of drugs to selectively activate signalling pathways and/or regulatory processes. These include biophysical measurement of GPCR-G protein coupling, direct assay of G protein activation and selective inhibition of G protein interaction with mini-genes. We are also applying molecular modelling and pharmacophore analysis to gain insight on functional groups that lead to specific receptor states.