Molecular Determinants of G Protein Signaling Selectivity

West Virginia University Research Corporation, Morgantown WV

Investigators

Abstract

On the order of a thousand different G protein coupled receptors (GPCR) exist in mammals to mediate a diverse array of physiological responses initiated by hormones, neurotransmitters, sensory stimuli, and other signaling molecules. Each GPCR is coupled to a heterotrimeric G protein that is critical for transmitting the signal initiated by receptor binding to the intracellular environment. A large number of distinct G protein heterotrimers are capable of forming from some 20 alpha, 6 beta and 12 gamma subunits. The abundance of distinct subtypes identified for each component is a striking feature of G protein coupled signaling pathways. The functional significance of such closely related molecules remains to be appreciated, and more importantly, the molecular basis for selective interactions among the components is largely unknown. At the biochemical level, significant contributions in this area can be made by developing the ability to employ components of signaling pathways in reconstitution paradigms. The ability to functionally couple receptors expressed in Sf9 cell membranes with exogenous G proteins has been developed and will be used to examine the G protein coupling behavior of distinct subtypes of muscarinic receptors capable of functionally distinguishing among individual G alpha subunits. The molecular basis of this selectivity will be defined by comparing the abilities of G protein heterotrimers containing chimeric alpha subunits, comprised of various regions of the alpha q subunit, to interact with the muscarinic M1 receptors. Functional interactions will be assessed by examining both the ability of the G protein to induce the high affinity state of the receptor and the ability of the agonist occupied receptor to catalyze guanine nucleotide exchange on the G protein. The unique feature of this project is the ability to examine interactions of defined molecular species in a single eukaryotic membrane environment using a reconstitution paradigm where the stoichiometries can be controlled with some precision. Identification of the individual amino acids on G alpha subunits involved in functional interactions with muscarinic receptors will be required to understand the selective interaction of these GPCRs with particular G alpha subunits and is likely to reveal the precise molecular mechanism underlying receptor mediation of the guanine nucleotide exchange process, the initial intracellular step in a profusion of signal transduction cascades.