Mutation of carboxyl-terminal threonine residues in human m3 muscarinic acetylcholine receptor modulates the extent of sequestration and desensitization.

We previously reported that a mutant human m3 muscarinic acetylcholine receptor in which threonine residues at positions 550, 553, and 554 in the carboxyl terminus had been substituted with alanines showed a significant blockage of receptor down-regulation when expressed in Chinese hamster ovary-K1 cells. Because Chinese hamster ovary cells showed little receptor sequestration, in the present study we investigated further the effects of these mutations on sequestration and desensitization in human embryonic kidney (HEK) 293 cells. Wild-type and mutant receptors were transiently transfected into HEK 293 cells. The level of m3 muscarinic acetylcholine receptor expression was approximately 300 fmol/mg protein, and the transfection efficiency was approximately 30% for all receptors. Also, wild-type and mutant receptors induced similar 4-fold increases in phosphoinositide (PPI) hydrolysis and showed similar Ca2+ responses after carbachol (CCh) treatment. However, the sequestration of wild-type receptors, determined as the difference between the extent of binding of lipophilic and hydrophilic ligands, occurred in a time- and dose-dependent manner to a maximum of approximately 40% of total receptors. In contrast, sequestration was almost totally blocked in cells expressing Ala550,553 or Ala550,553,554 mutant receptors. To determine the functional significance of sequestration and investigate its relationship to receptor desensitization, cells were preincubated with CCh and then washed free of agonist and restimulated with CCh. Desensitization was manifest as a time- and concentration-dependent decrease in the ability of the second stimulation to increase PPl hydrolysis. One-hour pretreatment with 1 mM CCh decreased PPl hydrolysis by 24% for wild-type receptors but had no effect on the ability of the mutant receptors to respond to a second CCh challenge. Furthermore, inhibition of wild-type receptor sequestration by treatment with conconavalin A also blocked desensitization to a 1-hr treatment with CCh. These results suggest that sequestration may be directly involved in m3 receptor desensitization at early times. More prolonged CCh treatment (3-9 hr) reduced the PPl hydrolysis response of the mutant and the wild-type receptors, indicating that the mechanism of m3 receptor desensitization at later times involves multiple components.