Differential agonist-induced regulation of human M2 and M3 muscarinic receptors

We have compared the regulation of M-2 and M-3 muscarinic receptors heterologously expressed in HEK-293 cells upon long-term exposure towards the agonist carbachol. Carbachol time- and concentration-dependently reduced M-2 receptor density with a maximum reduction of about 60%. Treatment with 1 mM carbachol for 24 hr 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 alpha-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 M-3 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 M-3 receptor was a reduction in the expression of the alpha-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 Ca2+ elevations by ATP and sphingosine-1-phosphate. Levels of M-2 and M-3 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 M-2 and M-3 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. (C) 2003 Elsevier Inc. All rights reserved.