We propose to continue our long-standing studies in the B-adrenergic receptor (BetaAR) signalling pathway of S49 lymphoma cells. These cells provide a unique model system in which to characterize the role of particular components in this pathway in which BAR increase cellular levels of cyclic AMP (cAMP), activate protein kinase A (PKA), and trigger events that ultimately lead to growth arrest and death of the cells. Feedback regulation of signalling occurs via a G protein receptor kinase (GRK), as well as PKA. Our preliminary studies indicate that cAMP-mediated cell death of S49 cells occurs via an apoptotic mechanism that requires PKA. Moreover, other recent findings implicate a key role of PKA desensitization of the BAR pathway. We propose to utilize wild-type S49 cells and known variants of S49 cells with lesions in the BAR response pathway (including an S49 cell line which does not undergo apoptosis in spite of apparently normal """"""""upstream"""""""" signalling components) to define the role of cAMP generation/PKA activation and mechanisms involved in apoptosis and in addition the contribution of G protein receptor kinase and PKA to ongoing feedback regulation of events involved in cAMP formation and action. Based on other preliminary results, we will also conduct studies of GRK and PKA using Chinese hamster ovary (CHO) cells, both wild-type and PKA-absent CHO cells. Data in the CHO system regarding receptor desensitization should complement studies in the S49 cell system. By using a combined approach that involves techniques of cell and molecular biology, biochemistry, and pharmacology, we should obtain new information regarding BAR signalling. Since BAR play an important physiologic role in many organ systems, in particular the cardiovascular, renal, and pulmonary systems, and BAR are targets of drug therapy in many clinically important disorders (e.g., hypertension, asthma, myocardial ischemia, chronic obstructive pulmonary disease, congestive heart failure), our results may provide useful new insights regarding both physiological regulation as well as drug therapy with agents that stimulate or block BetaAR signalling. Moreover, the results should prove applicable to the many other hormone and neurotransmitter receptors that link to G proteins.
