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

Abstract

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 αt (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 αq 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 αtL344A generated in αs and αi did not show such a severe loss of guanine nucleotide binding, revealing that the αt carboxyl terminus is unique in its susceptibility to changes in amino acid sequence. 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 αt (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 αq 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 αtL344A generated in αs and αi did not show such a severe loss of guanine nucleotide binding, revealing that the αt carboxyl terminus is unique in its susceptibility to changes in amino acid sequence.