Structure-Function Studies of GPCRs

Mutational analysis of allosteric binding sites on GPCRs

The crystal structure of bovine rhodopsin has been used as a template for homology modeling of other GPCRs, and has had some utility in predicting ligand-receptor interactions that occur within the transmembrane (TM) helices of certain GPCRs. For some GPCRs (e.g., the Family C GPCRs), the TM regions comprise an allosteric site, but for many others (e.g., aminergic receptors), they comprise the orthosteric pocket. We have recently been exploring the role of the extracellular loop regions of the M2 and M4 muscarinic acetylcholine receptors, which are far more variable between GPCR types thatn the TM regions, and have used mutagenesis to confirm the role of these extracellular loops in the binding of allosteric modulators to these receptors. In collaboration with Prof. Michael Parker and Dr. Craig Morton (St. Vincent’s Research Institute) and Prof. Ruben Abagyan (Scripps Research Institute), we have been applying a variety of computational approaches to develop predictive models of the allosteric sites on these receptors, with an aim to use these models to gain better insight into the structural basis of allosteric modulator selectivity.

Molecular modeling of the orthosteric binding site on Family B GPCRs

Modeling of Family B GPCRs provides an additional layer of complexity over work with Family A receptors due to the limited sequence conservation between Family B GPCRs and the only x-ray structure template (rhodopsin). To address this, we are collaborating with Prof. Miller (Mayo Clinic, Scottsdale, AZ, USA), Prof. Abagyan (Scripps Research Institute) and scientists at the software company Molsoft (La Jolla, CA) to develop novel methods for prediction of receptor structure. For this work, we also include information on ligand-receptor interaction derived from photoaffinity cross-linking, biophysical (FRET) and mutational experiments to derive constraints in the modeling process. We hope to develop predictive models of the receptors that may lead to new avenues for drug discovery.