FUNCTIONAL COUPLING OF Gi SUBTYPE WITH GABAB RECEPTOR/ADENYLYL CYCLASE SYSTEM: ANALYSIS USING A RECONSTITUTED SYSTEM WITH PURIFIED GTP-BINDING PROTEIN FROM BOVINE CEREBRAL CORTEX

doi:10.1016/S0197-0186(96)00138-6

Neurochemistry International

Volume 31, Issue 1, July 1997, Pages 21-25

https://doi.org/10.1016/S0197-0186(96)00138-6 Get rights and content

Abstract

A single molecular species of GTP-binding protein (G protein) has been purified from the bovine cerebral cortex. The immunoblot analysis indicated that the isolated G protein might be Gi₁ or Gi₂ but not Go, since it was reacted by specific antibodies, anti-Giα_1–2 and anti-Giα_1ν3, but not anti-Goα. When the Gi protein was reconstituted into phospholipid vesicles with partially purified GABA_B receptor and adenylyl cyclase, the stimulation of GABA_B receptor by its agonists induced the inhibition of forskolin-stimulated cAMP accumulation. This GABA-induced inhibition was abolished by CGP 55845A, an antagonist of GABA_B receptor. These results suggest that a Gi subtype, which was suggested to correspond to Gi₁ or Gi₂ may be functionally coupled with GABA_B receptor/adenylyl cyclase system. © 1997 Elsevier Science Ltd

Section snippets

Purification of GTP-binding protein

For the purification of a single molecular species of G protein, the bovine cerebral cortex (120 ∼ 160 g) was homogenized at 4°C in 10 mM Tris-HCl (pH 7.4) containing 10% sucrose with Ultra-Turrax homogenizer (Janke and Kunkel) according to the method of Sternweis and Robishaw (1984). The homogenate was centrifuged at 1400g for 10 min and the resulting supernatant was then centrifuged at 18,000g for 60 min. After washing with the same buffer, this pellet was suspended with TED buffer [20 mM

Identification of purified GTP-binding protein

The G protein was purified by DEAE-Sephacel, Ultrogel AcA34 and Mono Q column chromatographies. In order to identify the presence of G protein, the final single peak having [³⁵S]GTPγS binding activity was analysed using SDS-polyacrylamide gel electrophoresis and immunoblotting with subtype specific antibodies against G protein α subunits (Fig. 1). It showed the presence of several protein bands which might contain Giα (molecular weight: about 41 kDa) and Giβ (molecular weight: about 35 kDa).

DISCUSSION

In general, most G protein-coupled receptors, which inhibits an activity of adenylyl cyclase, couples to the Gi family. For example, in the case of D₂ receptor, the specificity of receptor-G protein interactions was examined in a reconstitution system with purified D₂ receptor and each Gi subtype such as Gi₁, Gi₂, Gi₃ (Senogles et al., 1990). The results indicated that the D₂ receptor preferred Gi₂ over the other Gi subtype, although the D₂ receptor was able to couple to three Gi subtypes,

Acknowledgements

This work was supported, in part, by Grant-in-Aid for Scientific Research and by International Scientific Research Program of the Japanese Ministry of Education, Science and Culture. We thank CIBA-GEIGY Co. Ltd. (Basel, Switzerland) for a kind gift of CGP 55845A.

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FUNCTIONAL COUPLING OF Gi SUBTYPE WITH GABAB RECEPTOR/ADENYLYL CYCLASE SYSTEM: ANALYSIS USING A RECONSTITUTED SYSTEM WITH PURIFIED GTP-BINDING PROTEIN FROM BOVINE CEREBRAL CORTEX

Abstract

Section snippets

Purification of GTP-binding protein

Identification of purified GTP-binding protein

DISCUSSION

Acknowledgements

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Biochem. Biophys. Res. Commun.

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FUNCTIONAL COUPLING OF Gi SUBTYPE WITH GABA_B RECEPTOR/ADENYLYL CYCLASE SYSTEM: ANALYSIS USING A RECONSTITUTED SYSTEM WITH PURIFIED GTP-BINDING PROTEIN FROM BOVINE CEREBRAL CORTEX