Optimized binding of [35S]GTPgammaS to Gq-like proteins stimulated with dopamine D1-like receptor agonists.
Subtypes of dopamine D1-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [35S]GTPgammaS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [35S]GTPgammaS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [35S]GTPyS binding reaction include protein concentration at 40 microg/ml, cationic concentrations of 120 mM Na+, 1.8 mM K+, and 20 mM Mg(2+), a molar guanine nucleotide ratio of 100,000 GDP to [35S]GTPgammaS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30-120 min. Under the optimized conditions, the D1-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [35S]GTPgammaS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [35S]GTPgammaS-bound proteins with specific Galpha antibodies showed that at least 70% of SKF38393-stimulated [35S]GTPgammaS binding was to Galphaq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Galphas and the adenylate cyclase pathway or to Galphaq and the phospholipase C pathway.
Panchalingam
S
S
Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, Baltimore 21201-1180, USA.
Undie
A S
AS
D000818:Animals; D003840:Deoxycholic Acid / Q000494:pharmacology; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism*; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism; D011869:Radioligand Assay; D051381:Rats; D017207:Rats, Sprague-Dawley; D017447:Receptors, Dopamine D1 / Q000819:agonists*
D008565:Membrane Proteins; D017447:Receptors, Dopamine D1; D003840:Deoxycholic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D020962:Heterotrimeric GTP-Binding Proteins