Opsin-G11-mediated signaling pathway for photic entrainment of the chicken pineal circadian clock.

Light is a major environmental signal for entrainment of the circadian clock, but little is known about the intracellular phototransduction pathway triggered by light activation of the photoreceptive molecule(s) responsible for the phase shift of the clock in vertebrates. The chicken pineal gland and retina contain the autonomous circadian oscillators together with the photic entrainment pathway, and hence they represent useful experimental models for the clock system. Here we show the expression of G11alpha, an alpha subunit of heterotrimeric G-protein, in both tissues by cDNA cloning, Northern blot, and Western blot analyses. G11alpha immunoreactivity was colocalized with pinopsin in the chicken pineal cells and also with rhodopsin in the outer segments of retinal photoreceptor cells, suggesting functional coupling of G11alpha with opsins in the clock-containing photosensitive tissues. The physical interaction was examined by coimmunoprecipitation experiments, the results of which provided evidence for light- and GTP-dependent coupling between rhodopsin and G11alpha. To examine whether activation of endogenous G11 leads to a phase shift of the oscillator, Gq/11-coupled m1-type muscarinic acetylcholine receptor (mAChR) was ectopically expressed in the cultured pineal cells. Subsequent treatment of the cells with carbamylcholine (CCh), an agonist of mAChR, induced phase-dependent phase shifts of the melatonin rhythm in a manner very similar to the effect of light. In contrast, CCh treatment induced no measurable effect on the rhythm of nontransfected (control) cells or cells expressing G(i/o)-coupled m2-type mAChR, indicating selectivity of the G-protein activation. Together, our results demonstrate the existence of a G11-mediated opsin-signaling pathway contributing to the photic entrainment of the circadian clock.