Dual Signaling of Human Mel1a Melatonin Receptors via Gi2, Gi3, and Gq/11 Proteins

Abstract

Mel 1a melatonin receptors belong to the super-family of guanine nucleotide-binding regulatory protein (G protein)-coupled receptors. So far, interest in Mel 1a receptor signaling has focused mainly on the modulation of the adenylyl cyclase pathway via pertussis toxin (PTX)-sensitive G proteins. To further investigate signaling of the human Mel 1a receptor, we have developed an antibody directed against the C terminus of this receptor. This antibody detected the Mel 1a receptor as a protein with an apparent molecular mass of approximately 60 kDa in immunoblots after separation by SDS-PAGE. It also specifically precipitated the 2-[125I]iodomelatonin (125I-Mel)-labeled receptor from Mel 1a-transfected HEK 293 cells. Coprecipitation experiments showed that G(i2), G(i3), and G(q/11) proteins couple to the Mel 1a receptor in an agonist-dependent and guanine nucleotide-sensitive manner. Coupling was selective since other G proteins present in HEK 293 cells, (G(i1), G(o), G(s), G(z), and G12) were not detected in receptor complexes. Coupling of the Mel 1a receptor to G(i) and G(q) was confirmed by inhibition of high-affinity 125I-Mel binding to receptors with subtype-selective G protein alpha-subunit antibodies. G(i2) and/or G(i3) mediated adenylyl cyclase inhibition while G(q/11) induced a transient elevation in cytosolic calcium concentrations in HEK 293 cells stably expressing Mel 1a receptors. Melatonin-induced cytosolic calcium mobilization via PTX-insensitive G proteins was confirmed in primary cultures of ovine pars tuberalis cells endogenously expressing Mel 1a receptors. In conclusion, we report the development of the first antibody recognizing the cloned human Mel 1a melatonin receptor protein. We show that Mel 1a receptors functionally couple to both PTX-sensitive and PTX-insensitive G proteins. The previously unknown signaling of Mel 1a receptors through G(q/11) widens the spectrum of potential targets for melatonin.

Chronomedicine and type 2 diabetes: shining some light on melatonin

“…Melatonin can also act through G i -coupled MT2 receptors in INS-1 cells to lower intracellular Ca 2+ levels [110,113], which is also predicted to reduce insulin secretion. Finally, melatonin can act through G q -coupled MT2 receptors to stimulate phospholipase C (PLC) and inositol triphosphate (IP3) activity [114], which is expected to boost insulin secretion. Together, these data suggest that melatonin can have both stimulatory and inhibitory influences on insulin secretion.…”

Melatonin receptors in pancreatic islets: good morning to a novel type 2 diabetes gene

“…MTNR1B, but not 1A, has been shown to inhibit formation of cGMP, via the soluble guanylate cyclase pathway [38]. In vitro, MTNR1A associates with G i2 and G i3 , which results in the inhibition of adenylate cyclase, and G q/11 , which activates the PLC pathway [39,40].…”

“…PKC, protein kinase C Activation of MTNR1B inhibits the formation of cGMP [38], and addition of melatonin to INS-1 cells decreases cellular levels of cGMP, paralleling the impairment of insulin secretion [61]. MTNR1A has also been implicated in signalling controlled by PLC [37], possibly via coupling to G q/11 [39]. It has been reported that stimulation of INS-1 cells with melatonin provokes the release of inositol 1,4, 5-triphosphate [58,62], and when G i coupling is blocked by pertussis toxin, a stimulatory effect of melatonin is uncovered [58].…”

The role of melatonin in diabetes: therapeutic implications

“…In addition, melatonin induced IP 3 liberation that allow Ca 2+ to flow into the cell from intracellular stores (51), a common mechanism that triggers insulin secretion by pancreatic β-cell. Alternatively, MT2-receptor-dependent signaling pathway of melatonin stimulates phospholipase C via Gq proteins, markedly elevating inositol triphosphate (IP 3 )/Ca 2+ from intracellular stores (52)(53)(54). The co-product of phospholipase C (PLC) activity, diacylglycerol (DAG), may lead to MAPK p38-modulated activation of protein kinase D (PKD), protein kinase C (PKC) and increased vesicle fusion ( Figure 2C).…”