The long term objective of this project is to relate adrenergic receptor (AR) molecular structure to hormone/drug interaction and receptor function. These receptors play key roles in regulating a host of physiologic processes in man. We propose to determine the structural components of the beta AR molecule which are involved in desensitization as deduced from site directed mutagenesis of the human beta2 AR gene. The sites chosen for alteration of the beta AR are located at intracellular regions of the molecule, and are suspected of being involved in one or more components of desensitization based on preliminary biochemical studies and their conserved nature throughout the family of G- protein coupled receptors. The """"""""uncoupling"""""""" of the agonist occupied phosphorylated form of the beta-adrenergic receptor from Gs appears to be augmented by an as yet undefined protein. Such a protein (arresting) has been found in the analogous rhodopsin transducing photoreceptor system. Thus, a second specific aim of this proposal is to identify and characterize this putative regulatory protein in the beta-adrenergic receptor system. Both the delineation of key structural domains of the beta AR associated with desensitization and the characterization of this new regulatory protein may afford the means of altering the clinically relevant phenomenon of desensitization. The alpha2 AR appears to have two or possibly three subtypes based on Southern blot hybridization studies using the platelet alpha2 AR gene as a probe. We propose to clone and sequence this third subtype and to express the receptor in stable cell lines. This will be followed by pharmacologic and biochemical characterization with the ultimate goal of providing a means of differentiating the alpha2 AR subtypes by ligand binding and functional studies. These structure-function relationships could offer the basis for development of therapeutic subtype specific agonists and antagonists.
Specific aims one and three will be carried out in this Physician Scientist Program during Phase I which also includes formal classroom studies at the Duke University Graduate School, while the second specific aim as listed above will be undertaken during Phase II.
