The inner medullary collecting duct of the mammalian nephron is responsible for the final concentration of urine via the stimulation of adenylyl cyclase (AC) in response to arginine vasopressin (AVP). This nephron segment also exhibits phospholipase C (PLC) activity when stimulated by epidermal growth factor (EGF). The studies proposed herein are aimed at further elucidating the mechanism of EGF signalling and examining the interactions between EGF and AVP signalling systems in cultured rat inner medullary collecting tubule (RIMCT) cells. Studies will be performed to examine the roles of EGF receptor autophosphorylation, the Ras:GAP complex, and of tyrosine phosphorylation of Gi in EGF-stimulated PIP2 hydrolysis. Substrates of the EGF receptor tyrosine kinase will be identified by immunoprecipitation with anti-phosphotyrosine followed by immunoblotting with substrate-specific antibodies. The effect of inhibition of EGF-RTK on 125I-EGF binding, 35S-GTPgammaS binding and EGF-stimulated GTPase activity will be examined. The mechanism of inhibition of EGF-stimulated PIP2 hydrolysis by protein kinase C (PKC) will be explored by examining 125I-EGF binding, 35S-GTPgammaS binding and EGF-stimulated GTPase activity in the absence or presence of activation of PKC. These studies will be performed in parallel in cells expressing the T654A mutant of the EGF receptor to assess the role of threonine 654 phosphorylation in PKC-mediated inhibition of EGF-stimulated PIP2 hydrolysis. Substrates of PKC will be identified by labeling cells with 32P-H3PO4, stimulating the kinase, and subjecting the tissue to immunoprecipitation and/or immunoblotting with appropriate antibodies. Similar studies will be performed to identify substrates of cAMP-dependent protein kinase (A-kinase) and to examine the effects of A- kinase activation on 125I-EGF binding, G-protein function and the activity of PLC. To determine the mechanism of inhibition of AVP-stimulated AC by PKC, the kinase will be stimulated directly by dioctanoylglycerol and potential effects of PKC on 125I-AVP receptor binding and AVP-stimulated GTPase activity will be examined. Finally, by infecting RIMCT cells with virus containing cDNAs coding for constitutive active and dominant negative mutants of pertussis toxin- (PT) insensitive G-alpha chains, I will perform studies aimed at determining which of these PT-insensitive alpha chains transduces alpha2-adrenergic inhibition of PGE2-stimulated AC activity in RIMCT cells.

R01DK043697

1992-02-01

1996-06-30