Differential agonist-induced regulation of human M₂ and M ₃ muscarinic receptors

We have compared the regulation of M₂ and M₃ muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time- and concentration- dependently reduced M₂ receptor density with a maximum reduction of about 60%. Treatment with 1mM carbachol for 24hr was accompanied by desensitisation of carbachol-induced Ca²⁺ elevations (maximum response reduced by 70%) but not by alterations in the expression of various G-protein α-subunits. Consistently, heterologous desensitisation of Ca²⁺ elevations by the purinergic receptor agonist ATP or by sphingosine-1-phosphate was not detected. In contrast, carbachol time- and concentration-dependently up-regulated M₃ receptors with maximum increases to about 350% of control values. The up-regulation was fully blocked by cycloheximide indicating that it was dependent on protein synthesis. Concomitant with the up-regulation of the M₃ receptor was a reduction in the expression of the α-subunit of G_q/11. The net effect of these two opposite regulatory mechanisms was a lack of alteration of carbachol-stimulated Ca²⁺ elevation. However, the reduction of G _q/11 was accompanied by a heterologous desensitisation of Ca ²⁺ elevations by ATP and sphingosine-1-phosphate. Levels of M ₂ and M₃ receptor mRNA as assessed by real-time PCR were not significantly altered by carbachol exposure for either receptor, suggesting that alterations of mRNA stability did not contribute to the observed changes in receptor number. We conclude that M₂ and M₃ receptor expression within the same cell undergoes differential agonist-induced regulation being accompanied by distinct regulation of G-protein expression leading to differential effects on signal transduction by other receptor systems.