DIFFERENTIAL ROLE OF THE CARBOXYL-TERMINAL TYROSINE IN DOWN-REGULATION AND SEQUESTRATION OF THE M2 MUSCARINIC ACETYLCHOLINE-RECEPTOR

Muscarinic acetylcholine receptor (mAChR) number can be altered in res ponse to sustained agonist exposure. Short term agonist exposure (seco nds to minutes) causes a rapid removal of mAChR from the cell surface (sequestration) while agonist exposure for longer periods of time (hou rs) causes a decrease in total receptor number (down-regulation). Tyro sine residues located in the cytoplasmic tails of a number of membrane receptors have been demonstrated to be important in the regulation by either sequestration, as is the case with the mannose 6-phosphate rec eptor and other receptors endocytosed via clathrin coated vesicles, or down-regulation, as is the case with the beta(2)-adrenergic receptor. Mutation of the lone cytoplasmic tail tyrosine residue (Tyr-459) of t he mammalian m2 mAChR to Phe, Trp, or Ala did not affect agonist-induc ed sequestration, although it significantly attenuated agonist-induced down-regulation. Conversion of m2 Tyr-459 to lie did not affect the r ate or extent of agonist-induced sequestration or down-regulation, but the sensitivity of this mutant receptor to agonist-induced down-regul ation was slightly decreased. Agonist and antagonist binding as well a s functional coupling to the inhibition of cAMP accumulation was unaff ected by any of the mutations to Tyr-459. These results are the first to identify a site in a mAChR involved in the down-regulation of recep tor in response to agonist.