Optimized Binding of [ super(35)S]GTP gamma S to Gq-Like Proteins Stimulated with Dopamine D sub(1)-Like Receptor Agonists

• Published in: Neurochemical research 2000-06, Vol.25 (6), p.759-767
• Main Authors: Panchalingam, S, Undie, A S
• Format: Article
• Language: English
• ISSN: 0364-3190
• DOI: 10.1023/A:1007553004615

Subtypes of dopamine D sub(1)-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 [ super(35)S]GTP gamma S binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [ super(35)S]GTP gamma S 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 [ super(35)S]GTP gamma S binding reaction include protein concentration at 40 mu g/ml, cationic concentrations of 120 mM Na super(+), 1.8 mM K super(+), and 20 mM Mg super(2+), a molar guanine nucleotide ratio of 100,000 GDP to [ super(35)S]GTP gamma S, the presence of 1 mM deoxycholate, and an overall incubation duration of 30-120 min. Under the optimized conditions, the D sub(1)-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [ super(35)S]GTP gamma S binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [ super(35)S]GTP gamma S-bound proteins with specific G alpha antibodies showed that at least 70% of SKF38393-stimulated [ super(35)S]GTP gamma S binding was to G alpha q. 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 G alpha s and the adenylate cyclase pathway or to G alpha q and the phospholipase C pathway.