Elsevier

Biochemical Pharmacology

Volume 56, Issue 10, 15 November 1998, Pages 1287-1293
Biochemical Pharmacology

Molecular and Cellular Pharmacology
Activation of various subtypes of G-protein α subunits by partial agonists of the adenosine A1 receptor

https://doi.org/10.1016/S0006-2952(98)00207-XGet rights and content

Abstract

ABSTRACT. The activation of different G protein subtypes by the rat adenosine A1 receptor initiated by stimulation with the full agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and by six structurally distinct partial agonists of this receptor was investigated. Endogenous G protein α subunits in rat cortical membranes were inactivated by N-ethylmaleimide (NEM). Activation of rat recombinant myristoylated αo, αi1, αi2 and αi3 by partial agonists in comparison to the full agonist was assessed by guanosine-5′-(γ-[35S]thio)triphosphate ([35S]GTPγS) binding after reconstitution of G protein α subunits with the adenosine A1 receptor in N-ethylmaleimide-treated membranes. 2-Chloro-N6-cyclopentyladenosine and 3′-deoxy-N6-cyclopentyladenosine (3′-d-CPA), the partial agonist with the highest intrinsic activity, were significantly more potent in activation of αi subtypes than αo. In contrast, 5′-methylthioadenosine (MeSA), 2′-deoxy-2-chloroadenosine (cladribine), 2′-deoxy-N6-cyclopentyladenosine (2′-d-CPA), 2-phenylaminoadenosine (CV 1808) and C8-aminopropyl-N6-cyclopentyladenosine (C8-aminopropyl-CPA) did not exhibit higher potency for Go or any Gi subtype. All partial agonists, although carrying structurally different modifications, showed higher relative intrinsic activities in activation of Gi than of Go, indicating that Gi-coupled pathways may be activated selectively via the A1 receptor by partial agonists, but not Go-mediated responses.

Section snippets

Materials

[35S]GTPγS (1,000–1,500 Ci/mmol) was obtained from New England Nuclear. Biologically active myristoylated rat recombinant G-protein α subunits (αo, αi1, αi2 and αi3) came from Calbiochem. Alamethicin, BSA, CHAPS, cladribine, GTPγS, MeSA and NEM were purchased from Sigma. Adenosine deaminase (from calf intestine; 200 U/mg), DTT and guanosine diphosphate were from Boehringer Mannheim. Polyethyleneglycol 6,000 was from Serva. CCPA and CV 1808 came from Research Biochemicals. C8-aminopropyl-CPA,

Results

Myristoylated rat recombinant G protein α subunits were reconstituted with adenosine A1 receptors in rat brain membranes after inactivation of endogenous α subunits by alkylation with NEM. In control membranes, CCPA induced an approximately two-fold increase in [35S]GTPγS binding over nonstimulated binding. Pretreatment of the membranes with NEM reduced the A1 receptor-dependent G protein activation from 1,391 ± 138 fmol/mg protein in control membranes to 60 ± 12 fmol/mg (4.3% of control; Fig. 1

Discussion

In order to characterize the activation of different subtypes of G-protein α subunits by agonists of the adenosine A1 receptor, we reconstituted individual rat recombinant G protein αo, αi1, αi2 and αi3 subunits with the adenosine A1 receptor in rat forebrain membranes after inactivation of the endogenous α subunits by alkylation of sulfhydryl groups with NEM. This treatment uncouples the receptor from G proteins, but does not affect the adenosine A1 receptor protein [25]. Similarly, Jockers et

Acknowledgements

We are grateful to Dr. A. P. IJzerman (Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands) for the generous gift of 2′-d-CPA, 3′-d-CPA and C8-aminopropyl-CPA. This study was supported by the European Commission through the BIOMED I concerted action ADEURO.

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