The effect of carboxyl-terminal mutagenesis of Gt alpha on rhodopsin and guanine nucleotide binding.
The carboxyl terminus of G protein alpha subunits plays an important role in receptor recognition. To identify the amino acids that participate in this interaction, COOH-terminal mutants of alpha t (the transducin alpha 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 beta 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 alpha q sequence (D346E/C347Y/G348N/F350V), in which guanine nucleotide binding was restored, implies that distinct motifs maintain the structure of the alpha 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 alpha tL344A generated in alpha s and alpha i did not show such a severe loss of guanine nucleotide binding, revealing that the alpha t carboxyl terminus is unique in its susceptibility to changes in amino acid sequence.
Osawa
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Department of Cell Biology and Anatomy, University of North Carolina at Chapel Hill 27599-7090, USA.
Weiss
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D000595:Amino Acid Sequence; D000818:Animals; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D017354:Point Mutation; D011485:Protein Binding; D012243:Rhodopsin / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D006150:Guanine Nucleotides; D012243:Rhodopsin; D019204:GTP-Binding Proteins