The G protein gamma subunit. Requirements for dimerization with beta subunits.
Guanine nucleotide-binding protein beta and gamma subunits form a tightly bound complex that can only be separated by denaturation. Assembly of beta and gamma subunits is a complicated process. The beta 1 and gamma 2 subunits can be synthesized in vitro in rabbit reticulocyte lysate and then assembled into dimers, but beta 1 cannot form beta gamma dimers when synthesized in a wheat germ extract. In contrast, gamma 2 translated in either system can dimerize with beta 1, suggesting that dimerization-competent gamma 2 can be synthesized without the aid of specific chaperonins or other cofactors. Dimerization-competent gamma 2 in solution forms an asymmetric particle with a Stokes radius of about 21 +/- 0.4 A (n = 4), s20,w of 0.9 S (range 0.8-1.0 S, n = 2), and frictional ratio of 1.57 (assuming no hydration). To define the part of gamma 2 that is needed for native beta gamma dimer formation, a series of N- and C-terminal truncations were generated, synthesized in vitro, and incubated with beta 1. Dimerization was assessed by stabilization of beta 1 to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization with beta 1, whereas removal of 27 amino acids prevented it. Therefore, a region between residues 45 and 59 of gamma 2 is important for dimerization. Truncation of 15 amino acids from the N terminus greatly diminished the formation of beta gamma dimers, while removal of 25 amino acids entirely blocked it. Thus, another region important for forming native beta gamma is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope did not prevent beta gamma dimerization. Furthermore, in intact 35S-labeled COS cells, epitope-tagged gamma 2 coimmunoprecipitates with beta and alpha subunits. The N-terminal epitope tag must lie at the surface of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody.
Mende
U
U
Cardiovascular Division, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Schmidt
C J
CJ
Yi
F
F
Spring
D J
DJ
Neer
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D000939:Epitopes; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism
D000939:Epitopes; D019204:GTP-Binding Proteins