We plan to characterize the association of G-proteins with their membrane bound receptors using experimental (including nuclear magnetic resonance, primarily transferred NOEs) and theoretical (including computer simulations for structure refinement and molecular modeling) techniques. The synthesis of peptides containing different cytoplasmic portions of the receptor has been partially realized and will be completed daring this granting period. The peptides will be examined in three different forms; linear- cyclized (utilizing a methylene linker to maintain a distance of 12 Angstroms between the termini, the same distance between the membrane spanning helices in bacteriorhodopsin) and cyclized with palmitoylated residues at both termini. The use of palmitoylated residues will facilitate the association of the peptide to the membrane. In this regard we have designed a peptide that should adopt a similar conformation to that found in the intact receptor. Each of the peptides will be tested for association and activation of the G-protein. The peptides will be conformationally examined both in aqueous solution and in the presence of micelles as a mimetic for a membrane environment. This portion of the project has been completed for two peptides. The next step is to characterize the conformational transition that the peptide undergoes upon association with the G-protein. Through the use of transferred NOEs the conformation of the small peptide while bound to the heterotrimeric G-protein (alpha, beta and gamma subunits) will be determined; experiments utilizing only the alpha-subunit have been planned. From the conformational features of the cytoplasmic loops of the receptor while free and bound to the G-protein the important constitutional and structural features far biological activity' will be elucidated. This insight will allow us to design molecules to regulate (enhance or inhibit) this important step in signal transduction. We are currently using the methods described above to examine the G- protein coupled parathyroid hormone receptor responsible for the maintenance of calcium levels and bone metabolism. The characterization of the interaction between this receptor and its related G-proteins (G-s, and G-q) will provide us with another target in the design of molecules to help in the control and regulation of calcium levels. The information obtained from these studies will be generally applicable for the characterization of the whole family of G-protein coupled receptors.