Serotonin 5-HT1A Receptor-mediated Erk Activation Requires Calcium/Calmodulin-dependent Receptor Endocytosis

Abstract

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-HT 1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrinmediated endocytosis. This activation was attenuated by chelation of intracellular Ca 2؉ and Ca 2؉/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, 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-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT 1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT 1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestrationdefective mutants of ␤-arrestin 1 and dynamin attenuated 5-HT 1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT 1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT 1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.Receptors coupled to heterotrimeric guanine nucleotidebinding (G) proteins, the largest known family of cell surface receptors, mediate cellular responses to many extracellular stimuli, such as neurotransmitters, peptide hormones, odorants, and photons (1). In addition to regulating the generation of soluble second messengers, many G protein-coupled receptors mediate proliferative or differentiative signals in various cultured cell lines and tissues via mitogen-activated protein (MAP) 1 kinases (2, 3). Erk1/2 MAP kinases are serine/threonine kinases involved in the activation of nuclear transcription factors. Erk1/2 activity is regulated by threonine/tyrosine phosphorylation, which is controlled by a highly conserved phosphorylation cascade. Phosphorylation of Erk1/2 is catalyzed by the MAP/Erk kinases 1 and 2 (MEK1 and MEK2) that are themselves phosphorylated and activated by MEK kinases such as raf-1 oncogene family proteins. Activation of Raf-1 occurs as a consequence of membrane translocation, which can be mediated by the GTP-bound form of the small G protein, Ras.Like the epidermal growth factor (EGF) receptor and other receptor tyrosine kinases, many G protein-coupled receptors regulate Ras function via tyrosine phosphorylation. Several G i -coupled receptors stimulate pertussis toxin-sensit...

G-Protein-Dependent Cell Surface Dynamics of the Human Serotonin1A Receptor Tagged to Yellow Fluorescent Protein

“…In addition, there appears to be a lack of consensus on the ability of ligands such as 8-OH-DPAT and 5-HT to induce internalization of 5-HT 1A receptors expressed in other cell types. Thus, while serotonin has been shown to mediate a modest degree (∼30%) of internalization of the hemagglutinin-epitope-tagged 5-HT 1A receptors in HEK-293 cells assessed using immunocytochemistry (52), studies carried out with 8-OH-DPAT using polyclonal antibodies against the receptor indicate that such a phenomenon occurs only for receptors in the neurons of the dorsal raphe nucleus but not in the hippocampus (53).…”

Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting

“…However, multiple signaling pathways can lead to the activation of ERK after stimulation of a particular GPCR, and some of these pathways may be activated independently of G proteins (for a review, see Werry et al, 2005). In the case of the 5-HT 1A R, the intracellular signaling pathway leading to ERK activation has been shown to implicate G ␤␥ subunits in CHO cells (Garnovskaya et al, 1996;Della Rocca et al, 1999). However, to date, it is not known whether the same pathway is involved in other cell lines, such as LLC-PK1 cells.…”

Physical Interaction of Calmodulin with the 5-Hydroxytryptamine2C Receptor C-Terminus Is Essential for G Protein-independent, Arrestin-dependent Receptor Signaling

“…Depending on the GPCR, CaM interaction with the receptor can affect its coupling with the G protein or its phosphorylation by protein kinase C (Nakajima et al, 1999;O'Connor et al, 1999;Wang et al, 1999;Bofill-Cardona et al, 2000;Turner et al, 2004;Turner and Raymond, 2005), suggesting a possible role in receptor desensitization. CaM can also directly contribute to receptor signaling (Della Rocca et al, 1999;O'Connor et al, 1999;Nickols et al, 2004). Notably, CaM has been involved in activation of ERK1,2 signaling pathway upon stimulation of certain GPCRs (Della Rocca et al, 1999;Belcheva et al, 2001).…”

“…The mechanisms by which CaM influences GPCR-dependent ERK1,2 signaling remain to be clarified and may differ from one GPCR to another. For example, the role of CaM in 5-HT 1A receptor-mediated ERK1,2 phosphorylation is critically dependent on its ability to promote receptor internalization (Della Rocca et al, 1999). Alternatively, CaM-dependent transactivation of the epidermal growth factor receptor has been involved in -opioid receptor-mediated ERK1,2 activation (Belcheva et al, 2001).…”

“…CaM can also directly contribute to receptor signaling (Della Rocca et al, 1999;O'Connor et al, 1999;Nickols et al, 2004). Notably, CaM has been involved in activation of ERK1,2 signaling pathway upon stimulation of certain GPCRs (Della Rocca et al, 1999;Belcheva et al, 2001). The mechanisms by which CaM influences GPCR-dependent ERK1,2 signaling remain to be clarified and may differ from one GPCR to another.…”