Re-evaluation of Adrenocorticotropic Hormone and Melanocyte Stimulating Hormone Activation of GPR139 in Vitro .

It is now well established that GPR139, a G-protein coupled receptor exclusively expressed in the brain and pituitary, is activated by the essential amino acids L-tryptophan (L-Trp) and L-phenylalanine (L-Phe) via G a q-coupling. The in vitro affinity and potency values of L-Trp and L-Phe are within the physiological concentration ranges of L-Trp and L-Phe. A recent paper suggests that adrenocorticotropic hormone (ACTH), a and b melanocyte stimulating hormones (alpha-MSH and beta-MSH) and derivatives a-MSH1 9/a-MSH1 10 can also activate GPR139 in vitro. We tested this hypothesis using guanosine 5'-O-(3-[S-35] thio)-triphosphate binding (GTP gamma S), calcium mobilization and [H-3] JNJ-63533054 radioligand binding assays. In the GTP gamma S binding assay, alpha-MSH, alpha-MSH1 9/alpha-MSH1 10, and beta-MSH had no effect on [S-35] GTP gamma S incorporation in cell membranes expressing GPR139 up to 30 m M in contrast to the concentration dependent activation produced by L-Trp, JNJ-63533054, and TC-09311 (two small molecule GPR139 agonists). ACTH slightly decreased the basal level of [S-35] GTP gamma S incorporation at 30 m M. In the GPR139 radioligand binding assay, a moderate displacement of [H-3] JNJ-63533054 binding by ACTH and b-MSH was observed at 30 m M (40 and 30%, respectively); alpha-MSH, alpha-MSH1 9/alpha-MSH1 10 did not displace any specific binding at 30 m M. In three different host cell lines stably expressing GPR139, alpha-MSH, and beta-MSH did not stimulate calcium mobilization in contrast to L-Trp, JNJ-63533054, and TC-09311. ACTH, alpha-MSH1 9/alpha-MSH1 10 only weakly stimulated calcium mobilization at 30 m M (< 50% of EC100). We then co-transfected GPR139 with the three melanocortin (MC) receptors (MC3R, MC4R, and MC5R) to test the hypothesis that ACTH, alpha-MSH, and b-MSH might stimulate calcium mobilization through a MCR/GPR139 interaction. All three MC peptides stimulated calcium response in cells co-transfected with GPR139 and MC3R, MC4R, or MC5R. The MC peptides did not stimulate calcium response in cells expressing MC3R or MC5R alone consistent with the Gs signaling transduction pathway of these receptors. In agreement with the previously reported multiple signaling pathways of MC4R, including Gq transduction pathway, the MC peptides produced a calcium response in cells expressing MC4R alone. Together, our findings do not support that GPR139 is activated by ACTH, alpha-MSH, and beta-MSH at physiologically relevant concentration but we did unravel an in vitro interaction between GPR139 and the MCRs.