The overall aim of this research is to understand the molecular basis of receptor-initiated signal transductions, primarily but not exclusively as they relate to adenylyl cyclase regulation. This includes cyclase stimulaton by Rs-type receptors and inhibition by Ri-type receptors. During the last grant period, we purified and characterized the subunit compositin of Ns and Ni, the coupling proteins that transduce Rs and Ri receptor occupancy into altered adenylyl cyclasle activity. We also described N-mediated regulation of hormone-receptor interactions and reconstituted RN interactions in phospholipid vesicles, using pure Rs and pure Ns and rhodopsin (a Ri analog) and pure Ni. We appear to have cloned the cDNA coding for the Mr = 35,000 beta subunit of N proteins, although final confirmation is still missing. During this coming grant period we wish: 1. To clone the cDNAs for the alpha and gamma subunits of Ns and Ni. 2. To express sn bacteria alpha, beta and gamma subunits of N proteins and study their respective functions as seen after recombination to give N proteins, using as assays the reconstitution in phospholipid vesicles of Rs-Ns and rhodopsin-Ni interactions. 3. To study the genetic organization of genes coding for Ns and Ni subunits and define the mode of biosynthesis of these proteins. 4. To explore if subunit dissociation occurs on receptor-mediated activation of N proteins in the presence of GTP and to determine how Ni acts, as seen after reconstitution in phospholipid vesicles of Ns, Ni, an Rs, an Ri and adenylyl cyclase proper. 5. To develop on the basis of possible sequence homologies between rhodopsin and Ri-type receptors an approach for cloning and determining the primary structure of Ri-type receptors such as alpha2-adrenergic, muscarinic acetylcholine and opioid receptors, or alternatively, to purify the muscarinic acetylcholine receptor and, on the basis of partial primary amino acid sequence determination or polyclonal antibody generation, to clone and determine its primary structure, define homologies with rhodopsin and proceed with cloning of other Ri-type receptors. 6. To explore possible structural homologies between N proteins and ras gene products testing their subunit composition and their possible physical coupling to receptors that do not couple to adenylyl cyclase. This research should significantly advance our understanding of the molecular basis of signal transductions that impinge on cAMP formation.
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