Calmodulin interacts with the third intracellular loop of the serotonin 5-hydroxytryptamine1A receptor at two distinct sites: putative role in receptor phosphorylation by protein kinase C

The serotonin 5-HT(1A) receptor couples to heterotrimeric G proteins and intracellular second messengers, yet no studies have investigated the possible role of additional receptor-interacting proteins in 5-HT(1A) receptor signaling. We have found that the ubiquitous Ca(2+)-sensor calmodulin (CaM) co-immunoprecipitates with the 5-HT(1A) receptor in Chinese hamster ovary fibroblasts. The human 5-HT(1A) receptor contains two putative CaM binding motifs, located in the N- and C-terminal juxtamembrane regions of the third intracellular loop of the receptor. Peptides encompassing both the N-terminal (i3N) and C-terminal (i3C) CaM-binding domains were tested for CaM binding. Using in vitro binding assays in combination with gel shift analysis, we demonstrated Ca(2+)-dependent formation of complexes between CaM and both peptides. We determined kinetic data using a combination of BIAcore surface plasmon resonance (SPR) and dansyl-CaM fluorescence. SPR analysis gave an apparent K(D) of approximately 110 nm for the i3N peptide and approximately 700 nm for the i3C peptide. Both peptides also caused characteristic shifts in the fluorescence emission spectrum of dansyl-CaM, with apparent affinities of 87 +/- 23 nm and 1.70 +/- 0.16 microm. We used bioluminescence resonance energy transfer to show that CaM interacts with the 5-HT(1A) receptor in living cells, representing the first in vivo evidence of a G protein-coupled receptor interacting with CaM. Finally, we showed that CaM binding and phosphorylation of the 5-HT(1A) receptor i3 loop peptides by protein kinase C are antagonistic in vitro, suggesting a possible role for CaM in the regulation of 5-HT(1A) receptor phosphorylation and desensitization. These data suggest that the 5-HT(1A) receptor contains high and moderate affinity CaM binding regions that may play important roles in receptor signaling and function.     

Physical interaction of calmodulin with the 5-hydroxytryptamine2C receptor C-terminus is essential for G protein-independent, arrestin-dependent receptor signaling

The serotonin (5-hydroxytryptamine; 5-HT)_2C receptor is a G protein-coupled receptor (GPCR) exclusively expressed in CNS that has been implicated in numerous brain disorders, including anxio-depressive states. Like many GPCRs, 5-HT_2C receptors physically interact with a variety of intracellular proteins in addition to G proteins. Here, we show that calmodulin (CaM) binds to a prototypic Ca²⁺-dependent “1-10” CaM-binding motif located in the proximal region of the 5-HT_2C receptor C-terminus upon receptor activation by 5-HT. Mutation of this motif inhibited both β-arrestin recruitment by 5-HT_2C receptor and receptor-operated extracellular signal-regulated kinase (ERK) 1,2 signaling in human embryonic kidney-293 cells, which was independent of G proteins and dependent on β-arrestins. A similar inhibition was observed in cells expressing a dominant-negative CaM or depleted of CaM by RNA interference. Expression of the CaM mutant also prevented receptor-mediated ERK1,2 phosphorylation in cultured cortical neurons and choroid plexus epithelial cells that endogenously express 5-HT_2C receptors. Collectively, these findings demonstrate that physical interaction of CaM with recombinant and native 5-HT_2C receptors is critical for G protein-independent, arrestin-dependent receptor signaling. This signaling pathway might be involved in neurogenesis induced by chronic treatment with 5-HT_2C receptor agonists and their antidepressant-like activity.     

A relationship between protein kinase C phosphorylation and calmodulin binding to the metabotropic glutamate receptor subtype 7.

Metabotropic glutamate receptor subtype 7 (mGluR7) is coupled to the inhibitory cyclic AMP cascade and is selectively activated by a glutamate analogue, L-2-amino-4-phosphonobutyrate. Among L-2-amino-4-phosphonobutyrate-sensitive mGluR subtypes, mGluR7 is highly concentrated at the presynaptic terminals and is thought to play an important role in modulation of glutamatergic synaptic transmission by presynaptic inhibition of glutamate release. To gain further insight into the intracellular signaling mechanisms of mGluR7, with the aid of glutathione S-transferase fusion affinity chromatography, we attempted to identify proteins that interact with the intracellular carboxyl terminus of mGluR7. Here, we report that calmodulin (CaM) directly binds to the carboxyl terminus of mGluR7 in a Ca(2+)-dependent manner. The CaM-binding domain is located immediately following the 7th transmembrane segment. We also show that the CaM-binding domain of mGluR7 is phosphorylated by protein kinase C (PKC). This phosphorylation is inhibited by the binding of Ca(2+)/CaM to the receptor. Conversely, the Ca(2+)/CaM binding is prevented by PKC phosphorylation. Collectively, these results suggest that mGluR7 serves to cross-link the cyclic AMP, Ca(2+), and PKC phosphorylation signal transduction cascades.     

Interaction of serotonin 5-hydroxytryptamine type 2C receptors with PDZ10 of the multi-PDZ domain protein MUPP1

By using the yeast two-hybrid system, we previously isolated a cDNA clone encoding a novel member of the multivalent PDZ protein family called MUPP1 containing 13 PDZ domains. Here we report that the C terminus of the 5-hydroxytryptamine type 2C (5-HT(2C)) receptor selectively interacts with the 10th PDZ domain of MUPP1. Mutations in the extreme C-terminal SSV sequence of the 5-HT(2C) receptor confirmed that the SXV motif is critical for the interaction. Co-immunoprecipitations of MUPP1 and 5-HT(2C) receptors from transfected COS-7 cells and from rat choroid plexus verified this interaction in vivo. Immunocytochemistry revealed an SXV motif-dependent co-clustering of both proteins in transfected COS-7 cells as well as a colocalization in rat choroid plexus. A 5-HT(2C) receptor-dependent unmasking of a C-terminal vesicular stomatitis virus epitope of MUPP1 suggests that the interaction triggers a conformational change within the MUPP1 protein. Moreover, 5-HT(2A) and 5-HT(2B), sharing the C-terminal EX(V/I)SXV sequence with 5-HT(2C) receptors, also bind MUPP1 PDZ domains in vitro. The highest MUPP1 mRNA levels were found in all cerebral cortical layers, the hippocampus, the granular layer of the dentate gyrus, as well as the choroid plexus, where 5-HT(2C) receptors are highly enriched. We propose that MUPP1 may serve as a multivalent scaffold protein that selectively assembles and targets signaling complexes.     

Agonist-induced phosphorylation of the serotonin 5-HT2C receptor regulates its interaction with multiple PDZ protein 1

Multiple PDZ domain protein 1 (MUPP1), a putative scaffolding protein containing 13 PSD-95, Dlg, ZO-1 (PDZ) domains, was identified by a yeast two-hybrid screen as a serotonin2C receptor (5-HT2C R)-interacting protein (Ullmer, C., Schmuck, K., Figge, A., and Lubbert, H. (1998) FEBS Lett. 424, 63-68). MUPP1 PDZ domain 10 (PDZ 10) associates with Ser458-Ser-Val at the carboxyl-terminal tail of the 5-HT2C R. Both Ser458 and Ser459 are phosphorylated upon serotonin stimulation of the receptor (Backstrom, J. R., Price, R. D., Reasoner, D. T., and Sanders-Bush, E. (2000) J. Biol. Chem. 275, 23620-23626). To investigate whether phosphorylation of these serines in the receptor regulates MUPP1 interaction, we used several approaches. First, we substituted the serines in the receptor carboxyl tail with aspartates to mimic phosphorylation (S458D, S459D, or S458D/S459D). Pull-down assays demonstrated that Asp mutations at Ser458 significantly decreased receptor tail interaction with PDZ 10. Next, serotonin treatment of 5-HT2C R/3T3 cells resulted in a dose-dependent reduction of receptor interaction with PDZ 10. Effects of serotonin on receptor-PDZ 10 binding could be blocked by pretreatment with a receptor antagonist. Alkaline phosphatase treatment reverses the effect of serotonin, indicating that agonist-induced phosphorylation at Ser458 resulted in a loss of MUPP1 association and also revealed a significant amount of basal phosphorylation of the receptor. We conclude that 5-HT2C R interaction with MUPP1 is dynamically regulated by phosphorylation at Ser458.     

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.     

Structural features underlying the unusual mode of calmodulin phosphorylation by protein kinase CK2: A study with synthetic calmodulin fragments

To shed light on the paradoxical behaviour of calmodulin, whose phosphorylation is inhibited by the regulatory beta-subunit of protein kinase CK2, a series of peptides encompassing the phosphoacceptor sites of calmodulin have been synthesized and assayed as substrates of CK2 alpha-subunit either alone or combined with the beta-subunit. The shortest peptide whose phosphorylation is reduced instead of being enhanced by the beta-subunit encompasses the sequence 68-106, including the central helix and the Ca2+-binding loop-III. In contrast, the phosphorylation of a peptide encompassing loop II and the central helix (54-92) is stimulated, like that of several shorter peptides, by the beta-subunit. Our data localize to the C-terminal domain of calmodulin the structural elements that are responsible for inverted susceptibility to beta-subunit regulation.     

Direct phosphorylation of the IL-2 receptor Tac antigen epitope by protein kinase C

Phorbol esters induce a rapid phosphorylation of the antigenic epitope of the human IL-2 receptor identified by anti-Tac monoclonal antibody. The physiological activator of protein kinase C, diacylglycerol also stimulated the phosphorylation of the Tac epitope in intact activated human T lymphocytes. Stable derivatives of cyclic nucleotides had no effect on the stimulation of Tac phosphorylation with cultured lymphocytes. Immunoprecipitated Tac derived from particulate membranes could serve as a direct substrate for purified protein kinase C in vitro. The Ca2+/phospholipid dependency of the in vitro phosphorylation reaction substantiated that the phosphorylation of Tac observed in intact cells stimulated by phorbol ester or diacylglycerol was the result of the physiological activation of protein kinase C.     

A Ca2+ calmodulin dependent kinase rather than protein kinase C is involved in up-regulation of the LHRH receptor.

Stimulation of protein kinase C (PKC) by phorbol ester (PMA) was reported previously to increase total binding of the peptide in whole rat pituitary cells. The effect could be obtained in cells from intact, not from spayed animals, suggesting a different level of spontaneous phosphorylation in both conditions. In the present work, endogenous PKC was desensitized in pituitary cells sampled from intact or 3 weeks castrated male rats and maintained in primary culture. Desensitization was induced by overnight incubation with 1 microM PMA. The maximum number of plasma membrane LHRH receptors (Bmax) present on cells from in intact animals was higher (+ 98 +/- 9%) when binding was performed at 0.5 degrees C instead of 21 degrees C as already observed in non PKC-desensitized cells. PMA (100 nM) was ineffective to increase Bmax, suggesting effectiveness of enzyme desensitization. In contrast, ionomycin 1 microM increased Bmax (53 +/- 10%). This increment was inhibited by W7, a calmodulin inhibitor, with an IC50 = 1 +/- 0.35 10(-6) M. No temperature dependency of the Bmax was observed in cells from castrated rats as already shown in the absence of PKC desensitization. Under these conditions, a Bmax decrease of 34 +/- 6% and 36.5 +/- 7.5% respectively was observed in the presence of H7, a PKC inhibitor, or of W7 (IC50 = 1 +/- 0.5 10(-5) M and IC50 = 0.8 +/- 0.2 10(-6) M). We conclude that a Ca2+ calmodulin dependent protein kinase rather than PKC itself is responsible for unmasking LHRH receptors.     

Interaction of the C-terminal region of the rat serotonin transporter with MacMARCKS modulates 5-HT uptake regulation by protein kinase C

The serotonin transporter (SERT) mediates the re-uptake of released serotonin into presynaptic nerve terminals. Its activity is regulated by different mechanisms including protein kinase C (PKC) triggered internalization. Here, we used yeast 2-hybrid screening and cotransfection into 293 cells to identify a homologue of the myristoylated alanine-rich C kinase substrate (MARCKS), MacMARCKS, as a C-terminally interacting protein of SERT. Upon cotransfection with SERT, MacMARCKS caused a reduction in the maximal rate of [(3)H]serotonin uptake and reduced its down-regulation elicited by activation of PKC. Our data are consistent with MARCKS proteins regulating the plasma membrane dynamics of neurotransmitter transporters.     

The in vitro phosphorylation of calmodulin by the insulin receptor tyrosine kinase

Calmodulin, a ubiquitous Ca2+-binding regulatory protein, is phosphorylated exclusively on tyrosine-99 in an insulin-dependent manner by wheat germ lectin-purified preparations of insulin receptors from rat adipocyte plasma membranes. Calmodulin is phosphorylated in the presence of polylysine, histone Hf2b, and protamine sulfate, but not in the absence of these cofactors or in the presence of other basic compounds known to interact with calmodulin, such as mellitin, myelin basic protein, chlorpromazine, trifluoperazine, substance P, glucagon, polyarginine, mastoparin, beta-endorphin, spermine, spermidine, and putrescine. The incorporation of 32P into calmodulin, expressed in terms of moles of phosphate per moles of calmodulin and assayed at calmodulin concentrations of 1.2 and 0.06 microM, is 0.023 + 0.002 and 0.046 + 0.006, respectively. This low stoichiometry is likely due to the relative impurity of the receptor preparation, as similar studies not shown here, using highly purified human insulin receptors, yield a stoichiometry of 1 mol phosphate/mol calmodulin. The time course of phosphorylation is characterized by a short initial lag phase of approximately 5 min, a rapid linear rate from approximately 5 to 40 min, with a steady state of 32P incorporation being approached at approximately 60 min. The K0.5 for ATP is 104 + 18 microM. Phosphorylated calmodulin is partially purified by HPLC on a C4 column using a trifluoroacetic acid/acetonitrile gradient solvent system. Phosphoamino acid analysis and limited thrombin digestion were used to determine that the site of insulin-induced phosphorylation of calmodulin is exclusively on tyrosine-99 regardless of the basic protein cofactor used. Phosphorylated calmodulin does not exhibit the characteristic Ca2+ shift normally observed with calmodulin in electrophoretic gels, an observation that is consistent with this modification affecting the biological activity of the molecule. Thus, the tyrosine phosphorylation of calmodulin represents a potentially important post-translational modification altering calmodulin's ability to regulate a variety of enzymes involved in growth, differentiation, and metabolic regulation.     

The insulin receptor and calmodulin. Calmodulin enhances insulin-mediated receptor kinase activity and insulin stimulates phosphorylation of calmodulin

Despite intensive research efforts, the functional role and regulation of the insulin receptor kinase remain enigmatic. In this investigation, we demonstrate that calmodulin enhances insulin-stimulated phosphorylation of the beta subunit of the insulin receptor and histone H2b and that insulin also stimulates phosphorylation of calmodulin. Using wheat germ lectin-enriched insulin receptor preparations obtained from rat adipocyte plasma membranes, calmodulin stimulated the rate and increased the amount of 32P incorporated predominantly into tyrosine residues of the beta subunit of the receptor when assayed in the presence of insulin. The stimulatory effect of calmodulin was both dose-dependent and saturable with half-maximal and maximal phosphorylation of the beta subunit occurring at 0.4 and 2.0 microM calmodulin, respectively. Ca2+ enhanced the ability of calmodulin to stimulate insulin-mediated phosphorylation of the beta subunit with an apparent K0.5 of approximately 0.6 microM. Calmodulin also induced an approximately 2-fold increase in both the rate and amount of insulin-mediated incorporation of 32P into histone H2b. The stimulatory effect of calmodulin was only observed in the presence of insulin and was concentration-dependent (K0.5 approximately 3.0 microM calmodulin), saturable (at 5 microM calmodulin), and Ca2+-dependent (K0.5 = 0.2 microM free Ca2+). Insulin also induced phosphorylation of a 17-kDa protein. On the basis of its molecular weight and purification via immunoadsorption with protein A-Sepharose-bound anti-calmodulin IgG, this phosphoprotein was identified as a phosphorylated form of calmodulin. Phosphorylation of calmodulin was only observed in the presence of insulin and was both Ca2+- and insulin concentration-dependent with half-maximal effects observed at 0.1 microM free Ca2+ and 350 microunits/ml insulin. Collectively, these results support the hypothesis that Ca2+ and calmodulin participate in the molecular mechanism whereby binding of insulin to its receptor is coupled to changes in cellular metabolism.     

Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension

Primary pulmonary hypertension is a progressive and often fatal disorder in humans that results from an increase in pulmonary blood pressure associated with abnormal vascular proliferation. Dexfenfluramine increases the risk of pulmonary hypertension in humans, and its active metabolite is a selective serotonin 5-hydroxytryptamine 2B (5-HT(2B)) receptor agonist. Thus, we investigated the contribution of the 5-HT(2B)receptor to the pathogenesis of pulmonary hypertension. Using the chronic-hypoxic-mouse model of pulmonary hypertension, we found that the hypoxia-dependent increase in pulmonary blood pressure and lung remodeling are associated with an increase in vascular proliferation, elastase activity and transforming growth factor-beta levels, and that these parameters are potentiated by dexfenfluramine treatment. In contrast, hypoxic mice with genetically or pharmacologically inactive 5-HT(2B)receptors manifested no change in any of these parameters. In both humans and mice, pulmonary hypertension is associated with a substantial increase in 5-HT(2B) receptor expression in pulmonary arteries. These data show that activation of 5-HT(2B) receptors is a limiting step in the development of pulmonary hypertension.     

Glycinergic ligands modulate the rate of phosphorylation of the glycine receptor by protein kinase C.

The alpha subunit of the glycine receptor purified from rat spinal cord is rapidly and specifically phosphorylated by protein kinase C (Ruiz-Gómez et al., (1991) J. Biol. Chem. 266, 559-566). We report here that the rate of phosphorylation of the glycine receptor by this kinase is higher in the presence of agonists (glycine, beta-alanine) than in the presence of antagonists (strychnine, RU-5135). These results suggest that activated glycine receptors would be a preferential target for functional regulation through phosphorylation mechanisms.     

Modulation of the G protein regulator phosducin by Ca2+/calmodulin-dependent protein kinase II phosphorylation and 14-3-3 protein binding

Phototransduction is a canonical G protein-mediated cascade of retinal photoreceptor cells that transforms photons into neural responses. Phosducin (Pd) is a Gbetagamma-binding protein that is highly expressed in photoreceptors. Pd is phosphorylated in dark-adapted retina and is dephosphorylated in response to light. Dephosphorylated Pd binds Gbetagamma with high affinity and inhibits the interaction of Gbetagamma with Galpha or other effectors, whereas phosphorylated Pd does not. These results have led to the hypothesis that Pd down-regulates the light response. Consequently, it is important to understand the mechanisms of regulation of Pd phosphorylation. We have previously shown that phosphorylation of Pd by cAMP-dependent protein kinase moderately inhibits its association with Gbetagamma. In this study, we report that Pd was rapidly phosphorylated by Ca(2+)/calmodulin-dependent kinase II, resulting in 100-fold greater inhibition of Gbetagamma binding than cAMP-dependent protein kinase phosphorylation. Furthermore, Pd phosphorylation by Ca(2+)/calmodulin-dependent kinase II at Ser-54 and Ser-73 led to binding of the phosphoserine-binding protein 14-3-3. Importantly, in vivo decreases in Ca(2+) concentration blocked the interaction of Pd with 14-3-3, indicating that Ca(2+) controls the phosphorylation state of Ser-54 and Ser-73 in vivo. These results are consistent with a role for Pd in Ca(2+)-dependent light adaptation processes in photoreceptor cells and also suggest other possible physiological functions.     

G protein-coupled receptor activation rapidly stimulates focal adhesion kinase phosphorylation at Ser-843. Mediation by Ca2+, calmodulin, and Ca2+/calmodulin-dependent kinase II

A rapid increase in the tyrosine phosphorylation of focal adhesion kinase (FAK) has been extensively documented in cells stimulated by multiple signaling molecules, but little is known about the regulation of FAK phosphorylation at serine residues. Stimulation of Swiss 3T3 cells with the G protein-coupled receptor agonists bombesin, vasopressin, or bradykinin induced an extremely rapid (within 5 s) increase in FAK phosphorylation at Ser-843. The phosphorylation of this residue preceded FAK phosphorylation at Tyr-397, the major autophosphorylation site, and FAK phosphorylation at Ser-910. Treatment of intact cells with ionomycin stimulated a rapid increase in FAK phosphorylation at Ser-843, indicating that an increase in intracellular Ca2+ concentration ([Ca2+]i) is a potential pathway leading to FAK-Ser-843 phosphorylation. Indeed, treatment with agents that prevent an agonist-induced increase in [Ca2+]i (e.g. thapsigargin or BAPTA (1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid)), interfere with calmodulin function (e.g. trifluoperazine, W13, and W7), or block Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation (KN93) or expression (small interfering RNA) abrogated the rapid FAK phosphorylation at Ser-843 induced by bombesin, bradykinin, or vasopressin. Furthermore, activated CaMKII directly phosphorylated the recombinant COOH-terminal region of FAK at a residue equivalent to Ser-843. Thus, our results demonstrate that G protein-coupled receptor activation induces rapid FAK phosphorylation at Ser-843 through Ca2+, calmodulin, and CaMKII.     

Requirement of protein association with membranes for phosphorylation by protein kinase C.

To clarify the requirement of the association of substrate proteins with phospholipid membranes for phosphorylation by protein kinase C (PKC), we studied the relationship between membrane association of PKC-substrate proteins and their phosphorylation by PKC. In the presence of phosphatidylserine, 12-O-tetradecanoylphorbol-13-acetate induced PKC autophosphorylation in either the presence or the absence of Ca2+, and this phosphorylation was not inhibited by increasing salt concentration (up to 200 mM NaCl). Thus, Ca2+ and ionic strength did not markedly affect the enzymatic activity of PKC. Annexin I required Ca2+ for both its association with phospholipid membranes and phosphorylation by PKC, whereas histone and monomyristilated lysozyme (C14:0-lysozyme) did not. This result indicates that the membrane association of substrates closely correlates with their phosphorylation by PKC. Similar correlation was also observed in the effects of ionic strength on the membrane association of the substrates and their phosphorylation by PKC; increased ionic strength (200 mM NaCl) remarkably inhibited both the membrane association and the phosphorylation of histone and annexin I by PKC but C14:0-lysozyme was not markedly affected. These results suggest that the membrane association of PKC-substrate proteins is a prerequisite for their phosphorylation by PKC. This concept further conforms to the mechanisms of PKC inhibitors; some types of PKC inhibitors are mediated all or in part through inhibition of the substrate-membrane interaction.