INTERACTIONS BETWEEN THE AMINO-TERMINAL AND CARBOXYL-TERMINAL REGIONSOF G-ALPHA SUBUNITS - ANALYSIS OF MUTATED G-ALPHA(0) G-ALPHA(I2) CHIMERAS/

Receptors activate the G alpha subunits of heterotrimeric G proteins b y binding to the C-terminus and reducing their affinity for bound GDP, therefore promoting exchange of GDP for GTP. Although this general me chanism is the same for all G alpha subunits, different G alpha subuni ts vary in nucleotide binding and hydrolysis even though the residues that make up the guanine nucleotide binding site are virtually identic al. We have shown previously that truncation of 14 amino acids from th e C-terminus of G alpha(o) decreased the apparent affinity for GDP and permitted us to see an activated conformation with GTP [Denker, B. M. , et al. (1992) J. Biol. Chem. 267, 9998-10002]. To test whether mutat ions in the receptor binding region lead to different phenotypes in cl osely related Ga subunits, we made the equivalent deletions in G alpha (i2), synthesized the proteins in vitro in a rabbit reticulocyte lysat e and used the pattern of native tryptic proteolysis as an index of co nformation. The phenotype of truncated G alpha(i2) was different from that of truncated G alpha(o): GDP affinity was reduced, but we could n ot detect an activated conformation with GTP (although GTP gamma S act ivated normally). Analysis of shorter deletions showed that loss of th ree hydrophobic residues (between 11 and 13 residues from the C-termin us) was responsible for the phenotypes. To define the regions of G alp ha(o) and G alpha(i2) that were responsible for their different phenot ypes, we used a conserved BamHI site (codon 212) to make chimeras. Eac h chimera truncated at the C-terminus had the phenotype of the donor o f the amino-terminal portion. Both truncated chimeras were activated b y GTP gamma S-like wild-type proteins, and both had decreased apparent affinity for GDP. Full-length chimeric subunits behaved like wild-typ e proteins. The crystal structure of G alpha(t) and G alpha(i1) shows that the three hydrophobic amino acids we have identified make contact with residues in the N- and C-terminal portions of the protein. Our s tudies point to the importance of the contacts in the N-terminal regio n (start of beta strands 1 and 3) that may stabilize the C-terminal al pha helix, affect nucleotide binding, and determine the characteristic features of different G alpha subunits.