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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033138-28
Application #
6525908
Study Section
Pharmacology A Study Section (PHRA)
Program Officer
Cole, Alison E
Project Start
1983-07-01
Project End
2005-08-31
Budget Start
2002-09-01
Budget End
2003-08-31
Support Year
28
Fiscal Year
2002
Total Cost
$341,925
Indirect Cost
Name
University of Wisconsin Madison
Department
Pharmacology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
Chu, Uyen B; Ramachandran, Subramaniam; Hajipour, Abdol R et al. (2013) Photoaffinity labeling of the sigma-1 receptor with N-[3-(4-nitrophenyl)propyl]-N-dodecylamine: evidence of receptor dimers. Biochemistry 52:859-68
Ruoho, Arnold E; Chu, Uyen B; Ramachandran, Subramaniam et al. (2012) The ligand binding region of the sigma-1 receptor: studies utilizing photoaffinity probes, sphingosine and N-alkylamines. Curr Pharm Des 18:920-9
Chu, Uyen B; Hajipour, Abdol R; Ramachandran, Subramaniam et al. (2011) Characterization of interactions of 4-nitrophenylpropyl-N-alkylamine with ýý receptors. Biochemistry 50:7568-78
Guo, Lian-Wang; Ruoho, Arnold E (2011) N-terminal half of the cGMP phosphodiesterase gamma-subunit contributes to stabilization of the GTPase-accelerating protein complex. J Biol Chem 286:15260-7
Mavlyutov, Timur A; Nickells, Robert W; Guo, Lian-Wang (2011) Accelerated retinal ganglion cell death in mice deficient in the Sigma-1 receptor. Mol Vis 17:1034-43
Guo, Lian-Wang; Hajipour, Abdol R; Ruoho, Arnold E (2010) Complementary interactions of the rod PDE6 inhibitory subunit with the catalytic subunits and transducin. J Biol Chem 285:15209-19
Zahmatkesh, Saeed; Hajipour, Abdol R (2010) Microwave-assisted synthesis and characterization of optically active poly (ester-imide)s incorporating L-alanine. Amino Acids 38:1253-60
Chu, Uyen B; Song, Jikui; Mavlyutov, Timur A et al. (2010) In vitro interaction of tubulin with the photoreceptor cGMP phosphodiesterase gamma-subunit. Neurosci Lett 482:225-9
Hajipour, Abdol R; Zahmatkesh, Saeed; Roosta, Parniyan et al. (2009) Synthesis and characterization of new optically active poly(azo-ester-imide)s via interfacial polycondensation. Amino Acids 36:511-8
Guo, Lian-Wang; Ruoho, Arnold E (2008) The retinal cGMP phosphodiesterase gamma-subunit - a chameleon. Curr Protein Pept Sci 9:611-25

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