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 i n the carboxyl terminus had been substituted with alanines showed a si gnificant blockage of receptor down-regulation when expressed in Chine se hamster ovary-K1 cells, Because Chinese hamster ovary cells showed little receptor sequestration, in the present study we investigated fu rther the effects of these mutations on sequestration and desensitizat ion in human embryonic kidney (HEK) 293 cells. Wild-type and mutant re ceptors were transiently transfected into HEK 293 cells. The level of m3 muscarinic acetylcholine receptor expression was similar to 300 fmo l/mg protein, and the transfection efficiency was similar to 30% for a ll receptors. Also, wild-type and mutant receptors induced similar 4-f old increases in phosphoinositide (PPI) hydrolysis and showed similar Ca2+ responses after carbachol (CCh) treatment. However, the sequestra tion 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 similar to 40% of tot al receptors. In contrast, sequestration was almost totally blocked in cells expressing Ala(550,553) Or Ala(550,553,554) mutant receptors. T o determine the functional significance of sequestration and investiga te its relationship to receptor desensitization, cells were preincubat ed with CCh and then washed free of agonist and restimulated with CCh, Desensitization was manifest as a time- and concentration-dependent d ecrease in the ability of the second stimulation to increase PPI hydro lysis. One-hour pretreatment with 1 mM CCh decreased PPI hydrolysis by 24% for wild-type receptors but had no effect on the ability of the m utant receptors to respond to a second CCh challenge. Furthermore, inh ibition of wild-type receptor sequestration by treatment with conconav alin A also blocked desensitization to a l-hr treatment with CCh. Thes e results suggest that sequestration may be directly involved in m3 re ceptor desensitization at early times. More prolonged CCh treatment (3 -9 hr) reduced the PPI hydrolysis response of the mutant and the wild- type receptors, indicating that the mechanism of m3 receptor desensiti zation at later times involves multiple components.