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