The focus of the proposed research is to understand the molecular interactions which regulate G protein-coupled receptor (GPCR) signaling through the use of multiple approaches. This work will involve the following Specific Aims: (1) To continue experiments to characterize the """"""""exosite"""""""" in the human b2AR using the newly developed I[125] iodoazidosalmeterol. This new photolabel is designed to covalently react with that portion of the b2AR, which interacts with the hydrophobic aryloxyalkyl tail which contributes to the long-acting property of salmeterol. Radiolabeled peptides will be generated from the photolabeled human b2AR, which is overexpressed in cultured HEK 293 cells. These peptides will be purified, N-terminal sequenced, and their position in the b2AR sequence determined. The precise derivatization sites will be determined by radiosequencing. (2) To identify interacting domains using """"""""tethered molecules."""""""" Development and use of photoactivatable derivatives of specific domain-interacting peptides, which will allow identification of intermolecular domains, which interact between the b2AR and Gas and between rhodopsin and a transducin (at). Chimeric proteins consisting of bacteriorhodopsin (BR) and intracellular loops of rhodopsin and the b2AR will be co-crystallized with holotransducin (BR/rhodopsin) and with Gs (BR/b2AR). Emphasis will be placed on the crystal structure of BR/i-3 loop chimeras initially with the cognate G proteins. (3) To characterize the interaction of the cGMP phosphodiesterase gammasubunit (PDEgamma) with the a subunit of transducin in the absence and presence of RGS-9/Gb5.
This Aim will involve the use of full-length PDEgamma photoaffinity labels containing benzophenones at various positions throughout the PDEgamma sequence. Using purified transducin and PDEgamma photolabels, crosslinked peptide sequences will be identified using mass spectroscopy and individual Gat residues identified using reversible benzophenone PDEgamma derivatives. Additional structural information will be obtained throught the use of NMR spectroscopy on the transducin bound form of PDEgamma. (4) To determine the role of synapse associated proteins (SAPs), in particular SAP97, in b2AR function.
This Aim i nvolves characterization of the interaction between SAPs and b2AR and identification of other partners, such as alpha-actinin, actin, and adenylyl cyclase type VI, in the signaling complex. This will increase our understanding of receptor-G protein-effector coupling systems, such as catecholamine b-receptors, which play a major role in the regulation of heart rate, blood pressure, heart failure, and other cardiovascular pathologies.
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