Two forms of the bovine brain G(o) that stimulate the inositol trisphosphate-mediated Cl- currents in Xenopus oocytes: Distinct guanine nucleotide binding properties

E. Padrell, D. J. Carty, T. M. Moriarty, J. D. Hildebrandt, E. M. Landau, R. 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 G(o) were identified. Both forms had stochiometric amounts of α- and βγ-subunits. The a-subunits of both forms were recognized by an α(o)-specific antiserum, but not by any of the α(i)-specific 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 α(o1). Both the recombinant α(o1) and the most abundant form of G(o) were recognized by an antiserum, H-660, against a peptide encoding amino acids 3-17 of α(i2). H-660 has been shown previously to recognize α(o) and α(i) (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 G(o) A most likely corresponds to the cloned α(o1). The less abundant form, G(o) B, was not recognized by H-660. However, both forms of bovine brain G(o) were recognized by GC/2, an antiserum against the N-terminal region of α(o1). Hence α(oA) and α(oB) may be different in their N terminus regions. Neither form of bovine brain G(o) was recognized by an antisera made to a peptide encoding the unique regions of the cloned α(o2) 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). G(o) A and G(o) 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 G(o) A binds GTPγS about 2.5-fold faster than G(o) B, in the absence of added Mg2+ ion. Both forms of G(o) as well as the recombinant α(o) (α(o1)) can increase muscarinic stimulation of inositol trisphosphate-mediated Cl- current in Xenopus oocytes. These data indicate that we have identified two structurally distinct forms of G(o) 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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