Regulation of melatonin 1a receptor signaling and trafficking by asparagine-124

Melatonin is a pineal hormone that regulates seasonal reproduction and has been used to treat circadian rhythm disorders. The melatonin 1a receptor is a seven- transmembrane domain receptor that signals predominately via pertussis toxin-sensitive G-proteins. Point mutations were created at residue N124 in cytoplasmic domain II of the receptor and the mutant receptors were expressed in a neurohormonal cell line. The acidic N124D- and E-substituted receptors had high-affinity (125)I-melatonin binding and a subcellular localization similar to the neutral N124N wild-type receptor. Melatonin efficacy for the inhibition of cAMP by N124D and E mutations was significantly decreased. N124D and E mutations strongly compromised melatonin efficacy and potency for inhibition of K(+)-induced intracellular Ca(++) fluxes and eliminated control of spontaneous calcium fluxes. However, these substitutions did not appear to affect activation of Kir3 potassium channels. The hydrophobic N124L and N124A or basic N124K mutations failed to bind (125)I-melatonin and appeared to aggregate or traffic improperly. N124A and N124K receptors were retained in the Golgi. Therefore, mutants at N124 separated into two sets: the first bound (125)I-melatonin with high affinity and trafficked normally, but with reduced inhibitory coupling to adenylyl cyclase and Ca(++) channels. The second set lacked melatonin binding and exhibited severe trafficking defects. In summary, asparagine-124 controls melatonin receptor function as evidenced by changes in melatonin binding, control of cAMP levels, and regulation of ion channel activity. Asparagine-124 also has a unique structural effect controlling receptor distribution within the cell.