Section 9
Chapter 8,078

A site on transducin alpha-subunit of interaction with the polycationic region of cGMP phosphodiesterase inhibitory subunit

Artemyev, N.O.; Mills, J.S.; Thornburg, K.R.; Knapp, D.R.; Schey, K.L.; Hamm, H.E.

Journal of Biological Chemistry 268(31): 23611-23615


ISSN/ISBN: 0021-9258
PMID: 8226888
Accession: 008077644

Download citation:  

Activation of cGMP phosphodiesterase (PDE) by the rod G-protein transducin is a key event in visual signal transduction in vertebrate photoreceptor cells. Interaction between the GTP-bound form of the alpha-subunit of transducin (alpha-t*) and the PDE inhibitory gamma-subunit (P-gamma) is a major component of PDE activation. The central polycationic region of P-gamma, P-gamma-24-45, has been implicated as one of the sites involved in alpha-t* cntdot P-gamma interaction. Here we determine the site on alpha-t* that interacts with P-gamma-24-45 using a photo-cross-linking approach. The synthetic peptides Cys(ACM)Tyr-P-gamma-24-45-Cys (where ACM indicates acetamidomethyl group) and Cys-P-gamma-24-45 were labeled with 4-(N-maleimido)benzophenone at the COOH and NH-2 termini, respectively, and then cross-linked to alpha-t. When the photoprobe was attached to the COOH terminus of the peptide, a specific high yield cross-linked product (80%) was formed between the peptide and alpha-tGTP-gamma-S (guanosine 5'-O-(thiotriphosphate)). A lower yield of cross-linking (35%) was seen between the peptide and alpha-tGDP. The site of cross-linking between Cys(ACM)Tyr-P-gamma-24-45-Cys and alpha-t GTP-gamma-S was localized to within alpha-t-306-310 using a variety of chemical and proteolytic cleavages of the cross-linked product, analysis of the fragments with SDS-polyacrylamide gel electrophoresis, and matrix-assisted laser desorption ionization mass spectrometry.

PDF emailed within 1 workday: $29.90