The goal of this proposal is to use a combination of biochemical and molecular biological approaches to determine the specificity and mechanism of G protein subunit interactions and interactions with opsins. The PI and his colleagues have developed several analytical techniques for analyzing the interacting domains and/or subtypes of transducin and other G protein alpha, beta and gamma subunits in the beta-gamma dimer, the alpha-beta-gamma trimer and the interactions between G protein trimers, subunits or peptides derived from subunits or opsins with the photoreceptor protein(s). There are five specific aims. The first is to identify the nature of the interaction of rhodopsin with Gt-alpha and gamma-1. The approaches used in this aim will be (1) alanine mutagenesis of hydrophobic vs charged residues in a C-terminal tail peptide of gamma-1 where the ability of substituted peptides to stabilize light-activated rhodopsin, metarhodopsin II, will be determined (2) structural analysis of wild-type and mutant peptides by 1D and NOESY NMR methods (3) making the corresponding mutations in recombinant gamma-1 to determine the effects of these substitutions on Gt heterotrimer interactions with rhodopsin and (4) using alpha-t and gamma-1 tail peptides and a mutant rhodopsin that binds Gt but does not activate it to determine if sequential interactions between rhodopsin and these two domains occurs.
The second aim i s to expand upon the initial observation by the PI that a conformational switch in beta-gamma unmasks the gamma-1 tail during receptor interaction. The approaches used in this aim are (1) to determine if mutations in gamma-1 can induce binding of beta-gamma to rhodopsin in the absence of alpha-t, (2) to use carboxypeptidase digestion of the gamma subunit as a probe to determine if the gamma-tail, masked in the beta-gamma complex is exposed upon interaction with rhodopsin, and (3) to use antibodies against different epitopes of the beta and gamma subunits to determine whether those epitopes are exposed prior to and/or during interaction with rhodopsin.
The third aim i s to use peptides specific to cytoplasmic loops of rhodopsin as competitors to determine whether these regions of the photoreceptor interact with the tails of alpha-t and gamma-1.
The fourth aim i s to test the hypothesis that different gamma subunits provide specificity of contact between receptors and G proteins. The two approaches that will be used for these studies are (1) to determine if peptides specific to different gamma subunits selectively interact with different rhodopsins (red, green and blue), using MII stabilization as an assay for effective interaction and (2) to determine if different recombinant heterotrimers composed of cone and rod photoreceptor specific alpha, beta and gamma subunits interact selectively with different opsins.
The final aim i s to use the yeast two-hybrid system to identify and isolate the specific beta subunits that associate with the gamma-c and-y subunits which have a potential role in visual signal transduction. Taken together these studies should provide new information concerning receptor-G protein subunit and subunit-subunit interacting domains, the mechanism of their interaction and may be useful in designing drugs that can modulate these types of interactions.
| Azpiazu, I; Gautam, N (2006) A G protein gamma subunit peptide stabilizes a novel muscarinic receptor state. Biochem Biophys Res Commun 341:904-10 |
| Kisselev, O G; Ermolaeva, M V; Gautam, N (1994) A farnesylated domain in the G protein gamma subunit is a specific determinant of receptor coupling. J Biol Chem 269:21399-402 |
| Pronin, A N; Gautam, N (1993) Proper processing of a G protein gamma subunit depends on complex formation with a beta subunit. FEBS Lett 328:89-93 |
| Kisselev, O; Gautam, N (1993) Specific interaction with rhodopsin is dependent on the gamma subunit type in a G protein. J Biol Chem 268:24519-22 |
