We have recently determined that several receptors functionally coupled to adenylyl cyclase can regulate Na-H exchange independently of changes in cAMP accumulation and independently of GTP-binding proteins previously described as being coupled to these receptors. Focusing on the Beta- adrenergic receptor (BetaAR), the objective of this proposal is to characterize the mechanisms whereby these receptors modulate Na-H exchange activity. Our current work indicates that two distinct pathways of receptor-regulated exchange may be occurring simultaneously. In cells expressing the ubiquitous NaH-1 exchanger, the BetaAR activates the exchanger protein through a GTP-dependent mechanism that does not involve Gs. In mutant fibroblasts lacking NaH-1, NaH-2 and NaH-3 proteins, however, an intrinsic ability of the receptor moiety itself to participate in a GTP-independent exchange of Na+ and H+ is indicated. With regard to the first pathway, we have determined that guanine nucleotides regulate Beta-adrenergic activation of the NaH-1 exchanger in cells expressing mutant constructs of the BetaAR that contain deletions of amino acid residues within the third cytoplasmic domain and are uncoupled from Gs. These findings indicate that distinct structural determinants of the receptor divergently regulate the Gs-dependent adenylyl cyclase pathway and the Gs-independent activation of the NaH-1 protein. Additionally, these findings suggest that the receptor is functionally coupled to the NaH-1 exchanger by a GTP-binding protein other than Gs. The goal of AIM 1 in this proposal is to identify structural properties of the BetaAR required for functional coupling to Na-H exchange by studying chimeric receptors and mutant BetaARs containing site-directed deletions.
AIM 2 seeks to isolate BetaAR-G protein complexes so as to characterize the regulatory protein that may couple the BetaAR to activation of the NaH-1 exchanger. The objective of AIM 3 is to further investigate the second pathway by determining whether the receptor protein has the capacity to act as a ligand-induced cation exchanger. We will study the ability of several receptors coupled to adenylyl cyclase to regulate an acid extrusion in mutant fibroblasts lacking antiporter proteins, and in conjunction with AIM 1, we will identify structural determinants of receptors required for this GTP- independent cation exchange. We will also study the ability of the purified BetaAR reconstituted into lipid vesicles to demonstrate a ligand-induced cation exchange. Although these objectives focus on the BetaAR, our studies indicate that the two independent mechanisms regulating Na-H exchange may be conserved by several receptors coupled to adenylyl cyclase. The research proposed in this application represents an examination of the molecular components of these pathways.
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