Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases , Erk1 and Erk2. The G(i)-coupled serotonin (5-hydroxytryptamine (5-HT)) 5- HT1A receptor, heterologously expressed in Chinese hamster ovary or human e mbryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechan ism dependent upon both Ras activation and clathrin-mediated endocytosis. T his activation was attenuated by chelation of intracellular Ca2+ and Ca2+/c almodulin (CAM) inhibitors or the CAM sequestrant protein calspermin, The C AM-dependent step in the Erk1/2 activation cascade is downstream of Ras act ivation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulat ed kinase). Inhibitors of the classical CAM effecters myosin light chain ki nase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phos phodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation . Because clathrin-mediated endocytosis was required for 5-HT1A receptor-me diated Erk1/2 activation, we pos tulated a role for CAM in receptor endocyt osis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin, and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist dependent endocytos is of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activ ation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosi s of the receptor, which is a required step in the activation of MEK and su bsequently Erk1/2.