The focus of this proposal is to understand the molecular interactions which occur in the various domains of the receptor- G protein-effector coupling system through the use of multiple approaches. Novel antagonist and agonist radioiodinated photoactive compounds which are selective for the catechol portion of the b2AR ligand binding site will be developed. These compounds will be used to photoaffinity label the ligand binding domain of the baculovirus-expressed Sf9 b2-AR. Partial N-terminal or complete sequence will be obtained for [125I] photolabeled peptides using a variety of photoprobes to """"""""map"""""""" the binding domain of the b2AR. The """"""""exosite"""""""" on the b2AR, which is involved in the mechanism of action of long-acting b2 agonists (i.e., salmeterol), will be identified using novel salmeterol derivatives. Soluble cytoplasmic domains of adenylyl cyclase will be expressed in E. coli and purified. The forskolin, ATP, and inhibitory """"""""p"""""""" site on the soluble adenylyl cyclase will be identified using photoactivatable compounds and purification of photolabeled peptides. Photolabeling experiments will also be performed on intact adenylyl cyclase, which will be overexpressed in Sf9 cells. Photoactivatable derivatives of specific domain-interacting peptides derived from the sequences of the b2AR and Gas will be utilized to identify interacting domains between b2AR and Gas, between Gas and adenylyl cyclase, and between rhodopsin and alpha transducin. The crystal structure of the catalytically active soluble IC1 and IIC2 adenylyl cyclase will be determined. The binding site domains for forskolin, nucleotides, and Gas will also be determined in the catalytically active soluble IC1 and IIC2 adenylyl cyclase by generating co-crystals. A component of this work involves expression and purification of large quantities of the IC1 domain and/or construction of catalytically active hybrid IC1. IIC2 molecules which are suitable for crystallography. These experiments will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine beta-receptors, which function to control autonomic functions, such as heart rate, blood pressure, and neuronal function and metabolic state of liver, adipose, and muscle.
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