The projects focus on examining the hypothesis that cell-cell signal transfer is an important factor in the regulation of cAMP-dependent protein kinases and by this means is influential in controlling cell growth and differentiation and numerous metabolic activities. There are five specific aims to be completed. 1) Complete the validation of an electron microscopic (EM) procedure for localizing free catalytic subunits (C) from the protein kinases and then establish an EM method for localizing free regulatory subunits (R) and the protein kinase inhibitor protein (PKI); 2) Evaluate the quantitative relationship between the amounts of hormone bound by ovarian granulosa cells and the resultant dissociation of R and C and activity of the PKI; 3) Determine whether or not cAMP is the signal transmitted through gap junctions; 4) Assess the effect of differentiation on the regulation of the protein kinase and the PKI by hormone-induced intercellular signal transfer; 5) Examine intercellular signal transfer between granulosa cells with defined populations of hormone receptors. These studies will be done using the flourescinated:PKI (F:PKI) prove for localizing intracellular sites of free C and a newly established flourescinated:C molecule that specifically complexes with the R subunit or the PKI in a cAMP-dependent manner. Also to be used is an 18 amino acid peptide, synthesized based on the sequence of a fragment of native PKI. The peptide is derivatized with flourophores or biotin and used as a stable substitute for F:PKI. Using these procedures and companion biochemical assays the studies will more fully test the regulation of cAMP-dependent protein kinases by hormone-induced signals transfer relative to its direct regulation by hormone binding to target cell receptors. The results will extend our understanding of how gap junction mediated communication is able to influence cell growth (including repair), differentiation, normal and abnormal, and various metabolic activities. From results obtained in this laboratory and elsewhere it has become increasingly clear that regulation of the cAMP-dependent protein kinases by hormone-induced signals, transmitted amongst cells via gap junctions, is a major influence in controlling normal cellular activity. Loss of this ability to exchange signals appears to coincide with neoplastic transformation that can lead to carcinome.
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