Structural and functional characterization of guanyl nucleotide-binding proteins using monoclonal antibodies to the alpha-subunit of transducin.
Transducin, the GTP-binding protein of the retinal light-sensitive phosphodiesterase system, and Gs and Gi, regulatory proteins of the hormone-sensitive adenylate cyclase, are members of a family of guanyl nucleotide-binding proteins termed G proteins that are important in signal transduction. To probe relationships within this family of G proteins, monoclonal antibodies were prepared against the alpha-subunit of bovine transducin (T alpha). Three of four monoclonal antibodies were specific for T alpha and did not cross-react with other G proteins. One, MAB1, cross-reacted strongly with the alpha-subunit of Gi (Gi alpha) purified from rabbit liver and, to a lesser extent, with the alpha-subunit of Go (Go alpha) purified from bovine brain and the proto-oncogene product H-ras p21. All four monoclonal antibodies recognized epitopes on a 23-kDa tryptic peptide fragment of T alpha which is derived from the N-proximal region. The three monoclonal antibodies that recognized only T alpha inhibited rhodopsin-stimulated GTP binding and hydrolysis by transducin, whereas MAB1 had no significant effect in these assays. These studies demonstrate that, within the 23-kDa tryptic peptide of T alpha, there is a domain(s) unique to T alpha that is involved in GTP binding and hydrolysis and another domain which is highly conserved in T alpha and to a lesser extent in other G proteins. Prior studies have identified regions involved in nucleotide binding and hydrolysis that are homologous in all G proteins. The observations reported here are consistent with the conclusion that the G proteins may have in addition unique regions involved in these functions.
J L
JL
Halpern
S C
SC
Tsai
R
R
Adamik
Y
Y
Kanaho
E
E
Bekesi
H F
HF
Kung
J
J
Moss
M
M
Vaughan
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000939:Epitopes / Q000032:analysis; D005260:Female; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D006868:Hydrolysis; D008565:Membrane Proteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D015293:Transducin
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D015293:Transducin