CAMP-dependent protein kinase and protein kinase C consensus site mutations of the beta-adrenergic receptor. Effect on desensitization and stimulation of adenylylcyclase
Yuan, N.; Friedman, J.; Whaley, B.S.; Clark, R.B.
Journal of Biological Chemistry 269(37): 23032-23038
Activation of cAMP-dependent protein kinase (cAPK) or protein kinase C (PKC) causes a rapid desensitization of beta 2-adrenergic receptor (beta AR) stimulation of adenylylcyclase in L cells, which previous studies suggest involves the cAPK/PKC consensus phosphorylation site in the third intracellular loop of the beta AR, RRSSK263. To determine the role of the individual serines in the cAPK- and PKC-mediated desensitizations, wild type (WT) and mutant beta ARs containing the substitutions, Ser261-->Ala, Ser262-->Ala, Ser262-->Asp, and Ser261/262-->Ala, were constructed and stably transfected into L cells. Results showed that serine 262 was the primary site of the cAPK-induced desensitization, whereas either serine 261 or serine 262 was sufficient to confer the 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA)/PKC-mediated desensitization. Coincident stimulation of cAPK and PKC caused an additive desensitization (6-8-fold increase in the EC50) which was significantly reduced (80%) only by the double substitution mutation. Quantitative evaluation of the coupling efficiencies and the GTP-shift of the WT and mutant receptors demonstrated that only one of the mutants, Ser262-->Ala, was partially uncoupled. The Ser262-->Asp mutation did not significantly uncouple, demonstrating that introducing a negative charge did not appear to mimic the desensitized state of the receptor. The beta AR expression level played a critical role in determining the pattern of beta AR desensitization; i.e. while the overall desensitization was unaltered within a large range of beta AR expression level (10-300 fmol/mg), the increase in EC50 and decrease in Vmax were differentially affected by the change in the receptor level.