Novel GPCR Screening Approach: Indirect Identification of S1P Receptor Agonists in Antagonist Screening Using a Calcium Assay

Abstract

To elucidate the physiological function of sphingosine 1-phosphate receptors 1–3 (S1P₁₋₃) we aimed to identify selective ligands for these GPCRs. S1P₂ and S1P₃ are coupled to G_q, and are, therefore, linked to the phospholipase C/IP₃/calcium pathway. S1P₁ is solely coupled to G_i and was artificially linked to calcium signaling by coexpression of Gα 16. The three receptors desensitized on challenge of cells with an agonist (i.e., agonists appeared as antagonists in a second calcium measurement). We screened a compound library for inhibitors of S1P-stimulated calcium signals, and we could identify agonists and antagonists with a single measurement. Agonism and antagonism were confirmed by recording compound-and S1P-induced calcium signals from the same assay well. For the three receptors, we found a reciprocal correlation of agonism and “apparent” antagonism of agonists. In addition, agonists indirectly discovered by this approach do not promote calcium mobilization through endogenous GPCRs.

Key Words: :

• G protein-coupled receptors
• Sphingosine 1-phosphate receptors
• Calcium measurements
• Receptor desensitization
• Apparent antagonism.