The Effect of Carboxyl-terminal Mutagenesis of G on Rhodopsin and Guanine Nucleotide Binding

• Published in: The Journal of biological chemistry 1995-12, Vol.270 (52), p.31052
• Main Authors: Shoji Osawa, Ellen R. Weiss
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.52.31052

The carboxyl terminus of G protein α subunits plays an important role in receptor recognition. To identify the amino acids that participate in this interaction, COOH-terminal mutants of α (the transducin α subunit) were expressed in vitro and analyzed for their ability to interact with rhodopsin and to bind guanine nucleotide. Gly-348, the reported site of a β turn, was replaced with other neutral amino acids without severely affecting rhodopsin binding. However, proline substitution abolished rhodopsin interaction, suggesting that flexibility is important at this site. A comparison between C347Y, which lost both rhodopsin and guanine nucleotide binding, and a mutant substituted with α sequence (D346E/C347Y/G348N/F350V), in which guanine nucleotide binding was restored, implies that distinct motifs maintain the structure of the α subunit and are necessary for selective interaction with receptors. Surprisingly, mutants L344A, L349A, F350stop, and stop351A demonstrated a parallel loss of rhodopsin and guanine nucleotide binding. Altered profiles of L344A and F350stop on sucrose density gradients indicate that these mutants may undergo denaturation. The equivalent of α L344A generated in α and α did not show such a severe loss of guanine nucleotide binding, revealing that the α carboxyl terminus is unique in its susceptibility to changes in amino acid sequence.