Two forms of the bovine brain Go that stimulate the inositol trisphosphate-mediated Cl- currents in xenopus oocytes: Distinct guanine nucleotide binding properties

Elena Padrell, Donna J. Carty, Thomas M. Moriarty, John D. Hildebrandt, Emmanuel M. Landau, Ravi Iyengar

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Abstract

Heterotrimeric GTP-binding proteins from bovine brain were resolved by fast protein liquid chromatography chromatography using Mono Q columns. Two distinct forms of the protein Go were identified. Both forms had stochiometric amounts of α- and βγ-subunits. The a-subunits of both forms were recognized by an αo-specific antiserum, but not by any of the arspecific antisera. The two forms showed distinct migration patterns on 9% sodium dodecyl sulfate-polyacrylamide gels containing 4-8 M urea gradients. Neither form comigrated with the recombinant αo1,. Both the recombinant αo1 and the most abundant form of Go were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of αi2. H-660 has been shown previously to recognize αo and αi (Mumby, S. M., Pang, I. K., Gilman, A. G., and Sternweis, P. C. (1988) J. Biol. Chem. 263, 2020-2026). This more abundant form is called Go A most likely corresponds to the cloned αo1- The less abundant form, Go B, was not recognized by H-660. However, both forms of bovine brain Go were recognized by GC/2, an antiserum against the N-terminal region of αo1- Hence αoA and αoB may be different in their N terminus regions. Neither form of bovine brain Go was recognized by an antisera made to a peptide encoding the unique regions of the cloned αo2 from HIT cells (Hsu W. H., Rudolph, U., Sanford, J., Bertrand, P., Olate, J., Nelson, C., Moss, L.E., Boyd, A. E., III, Codina, J., and Birnbaumer, L. (1990) J. Biol. Chem. 265, 11220-11226). Go A and Go B have similar guanine nucleotide binding and release properties. Both release GDP within 1 min in the absence of added Mg2+. Both bind guanosine (GTPγS) rapidly as well. However Go A binds GTPγS about 2.5-fold faster than Go B, in the absence of added Mg2+ ion. Both forms of Go as well as the recombinant αoo1) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl1 current in Xenopua oocytes. These data indicate that we have identified two structurally distinct forms of Go that have different guanine nucleotide binding properties and are capable of functioning in the receptor-regulated phospholipase C pathway in Xenopus oocytes.

Original languageEnglish
Pages (from-to)9771-9777
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number15
StatePublished - 1991

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