Sphingosine 1-phosphate: a Ca2+ release mediator in the balance.

Sphingosine 1-phosphate (S1P) is a lipid signalling molecule with Ca(2+) mobilising properties. Importantly for a role as a Ca(2+) release messenger, intracellular levels of S1P can be regulated by a variety of extracellular stimuli, via the enzyme sphingosine kinase. However, neither the mechanism underlying S1P generation, nor its actions at the endoplasmic reticulum are clear. Thus, the role of S1P as an intracellular mediator of Ca(2+) release remains in the balance.

Homologous and heterologous uncoupling of muscarinic M(3) and alpha(1B) adrenoceptors to Galpha(q/11) in SH-SY5Y human neuroblastoma cells.

1. The present study employed a [(35)S]-GTPgammaS binding protocol in conjunction with immunoprecipitation (IP) of the Galpha subunits to investigate the desensitization of G(q/11)-coupled receptors at the level of the G-protein activation. Membranes from SH-SY5Y cells expressing the recombinant human alpha(1B)-adrenoceptor (alpha(1B)-AR) (and endogenously expressing the M(3) muscarinic acetylcholine receptor (M(3)-AChR)) exhibited G(q/11) activation in a concentration-dependent manner in response to noradrenaline or methacholine. 2. Pre-treatment of intact cells with agonist prior to membrane preparation and use in the [(35)S]-GTPgammaS IP assay demonstrated that both receptors were homologously desensitized by pre-treatment with agonist since the G(q/11) activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of alpha(1B)-AR was effective at heterologously desensitizing the M(3)-AChR. The PKC inhibitor, Ro-31-8220 (10 microM) was ineffective at preventing the agonist-mediated receptor desensitization. 3. [(32)P]P(i)-labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 microM) was effective at producing a Ro-31-8220 (10 microM)-sensitive, detectable increase in alpha(1B)-AR but not M(3)-AChR phosphorylation. Noradrenaline (30 microM) stimulated alpha(1B)-AR phosphorylation, which could be partially inhibited by Ro-31-8220 (10 microM). The phosphorylation of M(3)-AChR was increased by methacholine (100 microM) incubation and this effect appeared to be insensitive to Ro-31-8220 (10 microM). 4. These findings demonstrate that [(35)S]-GTPgammaS-Galpha-subunit IP can be used to estimate receptor desensitization as a decline in receptor-G-protein coupling. Both the alpha(1B)-AR and M(3)-AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M(3)-AChR produced by alpha(1B)-AR stimulation is not associated with a detectable increase in M(3)-AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.

Inositol 1,4,5-trisphosphate-independent calcium signalling by platelet-derived growth factor in the human SH-SY5Y neuroblastoma cell.

In adherent SH-SY5Y human neuroblastoma cells, activation of G-protein-coupled muscarinic M3 receptors evoked a biphasic elevation of both intracellular [Ca(2+)] ([Ca(2+)]i) and inositol-1,4,5-trisphosphate (D-Ins(1,4,5)P3) mass. In both cases, temporal profiles consisted of rapid transient elevations followed by a decline to a lower, yet sustained level. In contrast, platelet-derived growth factor (PDGF), a receptor tyrosine kinase agonist acting via PDGF receptor b chains in these cells, elicited a slow and transient elevation of [Ca(2+)]i that returned to basal levels within 5 to 10 min with no evidence of inositol phosphate generation. Full responses for either receptor type required intracellular and extracellular Ca(2+) and mobilization of a shared thapsigargin-sensitive intracellular Ca(2+) store. Strategies that affected the ability of D-Ins(1,4,5)P3 to interact with the Ins(1,4,5)P3-receptor demonstrated an Ins(1,4,5)P3-dependency of the muscarinic receptor-mediated elevation of [Ca(2+)]i but showed that PDGF-mediated elevations of [Ca(2+)]i are Ins(1,4,5)P3-independent in these cells.

G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells.

We have investigated the effects of G protein-coupled receptor kinase (GRK) 3 and GRK6 on the phosphorylation and regulation of the M3 muscarinic acetylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexpression of GRK3 or GRK6 enhanced M3 mACh receptor phosphorylation after high-concentration methacholine (100 microM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 microM, 1 min) agonist stimulation. Compared with plasmid-transfected control cells expressing equivalent M3 mACh receptor number, GRK3- or GRK6-overexpressing cells exhibited a reduced phospholipase C activity reflected by a lower accumulation of total [3H]inositol phosphates and Ins(1,4,5)P3 mass. In addition, direct stimulation of G protein activation of phospholipase C (by AlF4(-)) was inhibited in GRK3- but not GRK6-overexpressing cells. Guanosine-5'-O-(3-[35S]thio)triphosphate binding and immunoprecipitation of Galpha(q/11) indicated that acute methacholine-stimulated receptor/Galpha(q/11) coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M3 mACh receptor uncoupling from Galpha(q/11), which was markedly enhanced by GRK6 overexpression, particularly at lower agonist pretreatment concentrations. However, the increased M3 mACh receptor phosphorylation seen in clones overexpressing GRK3 was not accompanied by increased receptor-Galpha(q/11) uncoupling. Overall, these data suggest that GRK3 and GRK6 use different pathways to desensitize the M3 mACh receptor. GRK6 seems to act as a classical GRK, inducing increased receptor phosphorylation accompanied by an uncoupling of receptor and Galpha(q/11). Conversely, GRK3 may cause desensitization independently of receptor phosphorylation, possibly via Gbetagamma binding and/or direct Galpha(q) binding via its regulator of G protein signaling domain to inhibit phospholipase C activity.

Enhanced inducible mGlu1alpha receptor expression in Chinese hamster ovary cells.

Inducible expression of the group-I metabotropic glutamate receptor (mGlu1alpha) in Chinese hamster ovary cells allows for the study of receptor density dependent effects. However, expression levels attainable with this system are lower than those reported for various brain regions and achieved by conventional (constitutive) transfection. Thus, direct comparison of mGlu1alpha receptor-mediated responses in this inducible expression system with those for receptors expressed heterologously or in vivo is compounded. We show here that inducible expression can be selectively augmented by butyrate pretreatment to levels approaching those reported for cerebral tissue. Enhanced mGlu1alpha receptor protein levels, agonist-induced inositol phosphate accumulation, as well as single-cell inositol 1,4,5-trisphosphate production and intracellular Ca(2+) mobilization occurred following co-induction with butyrate. In contrast, endogenous purinoceptor function was unaffected. Importantly, the ability to titrate receptor expression by varying isopropyl beta-thiogalactoside concentration was retained. Sodium butyrate thus offers a simple and convenient method to enhance inducible gene expression to levels found in vivo.

Single-cell imaging of graded Ins(1,4,5)P3 production following G-protein-coupled-receptor activation.

The pleckstrin homology domain of phospholipase Cdelta1 (PH(PLCdelta)) binds Ins(1,4,5)P(3) and PtdIns(4,5)P(2) specifically, and can be used to detect changes in Ins(1,4,5)P(3) in single cells. A fusion construct of PH(PLCdelta) and enhanced green fluorescent protein (EGFP-PH(PLCdelta)) associates with the plasma membrane due to its association with PtdIns(4,5)P(2). However, PH(PLCdelta) has greater affinity for Ins(1,4,5)P(3) than PtdIns(4,5)P(2), and translocates to the cytosol as Ins(1,4,5)P(3) levels rise. Prolonged activation of group I metabotropic glutamate receptor 1alpha expressed in Chinese-hamster ovary cells or endogenous M(3) muscarinic receptors in SH-SY5Y neuroblastoma cells gave an initial transient peak in translocation, followed by a sustained plateau phase. This closely followed changes in cell population Ins(1,4,5)P(3) mass, but not PtdIns(4,5)P(2) levels, which decreased monophasically, as determined by radioreceptor assay. Translocation thus provides a real-time method to follow increases in Ins(1,4,5)P(3). Graded changes in Ins(1,4,5)P(3) in Chinese-hamster ovary-lac-mGlu1alpha cells could be detected with increasing glutamate concentrations, and dual loading with fura 2 and EGFP-PH(PLCdelta) showed that changes in intracellular Ca(2+) concentration closely paralleled Ins(1,4,5)P(3) production. Moreover, Ins(1,4,5)P(3) accumulation and intracellular Ca(2+) mobilization within single cells is graded in nature and dependent on both agonist concentration and receptor density.

Cell type-specific differences in the coupling of recombinant mGlu1alpha receptors to endogenous G protein sub-populations.

In this study the effects of cell background on the coupling of the type 1alpha metabotropic glutamate (mGlu1alpha) receptor to different G protein sub-populations by recombinant expression of this receptor subtype in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells have been investigated. Receptor-G protein interactions were assessed using [(35)S]GTPgammaS binding and subsequent Galpha subunit-specific immunoprecipitation. In a CHO cell line (CHO-lac-mGlu1alpha), where mGlu1alpha receptor expression is under inducible control, stimulation of membranes with the mGlu receptor agonist quisqualate resulted in an increase in specific [(35)S]GTPgammaS binding to G(q/11)alpha only, whereas in a BHK cell line (BHK-mGlu1alpha) agonist stimulation increased [(35)S]GTPgammaS binding to G(q/11)alpha and also to pertussis toxin (PTx)-sensitive G(i/o) proteins (assessed using G(i1/2)alpha- and G(i3/o)alpha-specific antibodies). These data are consistent with our previous observations of dual, antagonistic G(q/11)/G(i/o) regulation of phospholipase C (PLC) in BHK-mGlu1alpha cells, whereas no evidence was found for a G(i/o) modulation of PLC activity in the CHO-lac-mGlu1alpha cell line. PTx pre-treatment of either cell line had no effect on either the magnitude or the concentration-dependency of agonist-stimulated [(35)S]GTPgammaS-G(q/11)alpha binding, excluding the possibility that receptor-G(i/o) uncoupling can unmask an increase in receptor-G(q/11) interaction. mGlu1alpha receptor expression per se had little effect on Galpha protein expression levels in either CHO or BHK cell lines, with the possible exception of a small, but consistent increase in G(o)alpha expression in BHK-mGlu1alpha cells compared to the vector-transfected control cell line (BHK-570). Semi-quantitative assessment of mGlu1alpha receptor immunoreactivity and [(3)H]quisqualate saturation binding analysis demonstrated a ca 10-fold higher mGlu1alpha receptor content in BHK cells. Whether the higher receptor expression level in BHK-mGlu1alpha cells underlies the additional G(i/o) coupling observed in this cell line, or additional factors contribute to the phenomenon are discussed.

Reassessment of the Ca2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurement of inositol 1,4,5-trisphosphate and Ca2+ in single cells.

Transient transfection of Chinese hamster ovary or baby hamster kidney cells expressing the Group I metabotropic glutamate receptor mGlu1alpha with green fluorescent protein-tagged pleckstrin homology domain of phospholipase Cdelta1 allows real-time detection of inositol 1,4,5-trisphosphate. Loading with Fura-2 enables simultaneous measurement of intracellular Ca(2+) within the same cell. Using this technique we have studied the extracellular calcium sensing property of the mGlu1alpha receptor. Quisqualate, in extracellular medium containing 1.3 mm Ca(2+), increased inositol 1,4,5-trisphosphate in all cells. This followed a typical peak and plateau pattern and was paralleled by concurrent increases in intracellular Ca(2+) concentration. Under nominally Ca(2+)-free conditions similar initial peaks in inositol 1,4,5-trisphosphate and Ca(2+) concentration occurred with little change in either agonist potency or efficacy. However, sustained inositol 1,4,5-trisphosphate production was substantially reduced and the plateau in Ca(2+) concentration absent. Depletion of intracellular Ca(2+) stores using thapsigargin abolished quisqualate-induced increases in intracellular Ca(2+) and markedly reduced inositol 1,4,5-trisphosphate production. These data suggest that the mGlu1alpha receptor is not a calcium-sensing receptor because the initial response to agonist is not sensitive to extracellular Ca(2+) concentration. However, prolonged activation of phospholipase C requires extracellular Ca(2+), while the initial burst of activity is highly dependent on Ca(2+) mobilization from intracellular stores.

G(q/11) and G(i/o) activation profiles in CHO cells expressing human muscarinic acetylcholine receptors: dependence on agonist as well as receptor-subtype.

1. Profiles of G protein activation have been assessed using a [35S]-GTPgammaS binding/immunoprecipitation strategy in Chinese hamster ovary cells expressing either M1, M2, M3 or M4 muscarinic acetylcholine (mACh) receptor subtypes, where expression levels of M1 and M3, or M2 and M4 receptors were approximately equal. 2. Maximal [35S]-GTPgammaS binding to G(q/11)alpha stimulated by M1/M3 receptors, or G(i1-3)alpha stimulated by M2/M4 receptors occurred within approximately 2 min of agonist addition. The increases in G(q/11)alpha-[35S]-GTPgammaS binding after M1 and M3 receptor stimulation differed substantially, with M1 receptors causing a 2-3 fold greater increase in [35S]-GTPgammaS binding and requiring 5 fold lower concentrations of methacholine to stimulate a half-maximal response. 3. Comparison of M2 and M4 receptor-mediated G(i1-3)alpha-[35S]-GTPgammaS binding also revealed differences, with M2 receptors causing a greater increase in G(i1-3)alpha activation and requiring 10 fold lower concentrations of methacholine to stimulate a half-maximal response. 4. Comparison of methacholine- and pilocarpine-mediated effects revealed that the latter partial agonist is more effective in activating G(i3)alpha compared to G(i1/2)alpha for both M2 and M4 receptors. More marked agonist/partial agonist differences were observed with respect to M1/M3-mediated stimulations of G(q/11)alpha- and G(i1-3)alpha-[35S]-GTPgammaS binding. Whereas coupling to these Galpha subclasses decreased proportionately for M1 receptor stimulation by these agonists, pilocarpine possesses a greater intrinsic activity at M3 receptors for G(i)alpha versus G(q/11)alpha activation. 5. These data demonstrate that mACh receptor subtype and the nature of the agonist used govern the repertoire of G proteins activated. They also provide insights into how the diversity of coupling can be pharmacologically exploited, and provide a basis for a better understanding of how multiple receptor subtypes can be differentially regulated.

Characterization of the metabotropic glutamate receptors mediating phospholipase C activation and calcium release in cerebellar granule cells: calcium-dependence of the phospholipase C response.

In this study we have determined the metabotropic glutamate receptors (mGluRs) involved in the glutamate activation of phospholipase C (PLC) and Ca(2+) mobilization in cerebellar granule cells at 9 days in vitro; and studied the Ca(2+) modulation of the PLC response. Both PLC activation and Ca(2+) signalling were found to be mediated exclusively by the mGluR1 subtype, although both group I mGluRs, mGluR1 alpha and mGluR5, could be detected in cell extracts. Exposure of cells to medium devoid of Ca(2+) for various times before agonist stimulation reduced the PLC response, which was quickly recovered following the re-exposure of cells to Ca(2+)-containing medium. The extent of the glutamate response correlated well with changes in the cytosolic Ca(2+) concentration. On the other hand, loading of the intracellular Ca(2+) stores by a transient depolarization followed by washing in nondepolarizing buffer, allowed glutamate to release stored Ca(2+) in the majority of cells and enhanced glutamate activation of PLC. Under such conditions, the absence of extracellular Ca(2+) during stimulation and the chelation of cytosolic Ca(2+) with BAPTA/AM inhibited both glutamate-elicited Ca(2+) response and PLC activation. Overall, these results indicate that the mGluR-mediated activation of PLC depends on the presence of extracellular Ca(2+) and can be modulated by moderate changes of cytosolic Ca(2+). Furthermore, ryanodine reduced PLC stimulation by glutamate in predepolarized cells but not in control cells, suggesting that ryanodine receptors could play a role in the potentiation of the mGluR-mediated activation of PLC by Ca(2+) release in predepolarized cells.

Intracellular sphingosine 1-phosphate production: a novel pathway for Ca2+ release.

Sphingolipids such as sphingosine 1-phosphate (SPP) and sphingosylphosphorylcholine have long been recognized to possess Ca2+ mobilizing activity, yet to date little is known about their mechanism of action, or indeed their significance as Ca2+ mobilizing intracellular messengers. The recent discovery of extracellular receptors for the sphingolipids has further complicated the interpretation of many experiments in this field. This paper reviews the current literature in which molecular and pharmacological approaches have begun to uncover the signalling components associated with intracellular SPP production and Ca2+ mobilization. The functional significance of this novel Ca2+ release pathway is also discussed.

Rapid down-regulation of the type I inositol 1,4,5-trisphosphate receptor and desensitization of gonadotropin-releasing hormone-mediated Ca2+ responses in alpha T3-1 gonadotropes.

Despite no evidence for desensitization of phospholipase C-coupled gonadotropin-releasing hormone (GnRH) receptors, we previously reported marked suppression of GnRH-mediated Ca(2+) responses in alphaT3-1 cells by pre-exposure to GnRH. This suppression could not be accounted for solely by reduced inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) responses, thereby implicating uncoupling of Ins(1,4,5)P(3) production and Ca(2+) mobilization (McArdle, C. A., Willars, G. B., Fowkes, R. C., Nahorski, S. R., Davidson, J. S., and Forrest-Owen, W. (1996) J. Biol. Chem. 271, 23711-23717). In the current study we demonstrate that GnRH causes a homologous and heterologous desensitization of Ca(2+) signaling in alphaT3-1 cells that is coincident with a rapid (t((12)) < 20 min), marked, and functionally relevant loss of type I Ins(1,4,5)P(3) receptor immunoreactivity and binding. Furthermore, using an alphaT3-1 cell line expressing recombinant muscarinic M(3) receptors we show that the unique resistance of the GnRH receptor to rapid desensitization contributes to a fast, profound, and sustained loss of Ins(1,4,5)P(3) receptor immunoreactivity. These data highlight a potential role for rapid Ins(1,4,5)P(3) receptor down-regulation in homologous and heterologous desensitization and in particular suggest that this mechanism may contribute to the suppression of the reproductive system that is exploited in the major clinical applications of GnRH analogues.

Lysophosphatidic acid-induced Ca2+ mobilization requires intracellular sphingosine 1-phosphate production. Potential involvement of endogenous EDG-4 receptors.

Lysophosphatidic acid (LPA)-mediated Ca(2+) mobilization in human SH-SY5Y neuroblastoma cells does not involve either inositol 1,4, 5-trisphosphate (Ins(1,4,5)P(3))- or ryanodine-receptor pathways, but is sensitive to inhibitors of sphingosine kinase. This present study identifies Edg-4 as the receptor subtype involved and investigates the presence of a Ca(2+) signaling cascade based upon the lipid second messenger molecule, sphingosine 1-phosphate. Both LPA and direct G-protein activation increase [(3)H]sphingosine 1-phosphate levels in SH-SY5Y cells. Measurements of (45)Ca(2+) release in premeabilized SH-SY5Y cells indicates that sphingosine 1-phosphate, sphingosine, and sphingosylphosphorylcholine, but not N-acetylsphingosine are capable of mobilizing intracellular Ca(2+). Furthermore, the effect of sphingosine was attenuated by the sphingosine kinase inhibitor dimethylsphingosine, or removal of ATP. Confocal microscopy demonstrated that LPA stimulated intracellular Ca(2+) "puffs," which resulted from an interaction between the sphingolipid Ca(2+) release pathway and Ins(1,4,5)P(3) receptors. Down-regulation of Ins(1,4,5)P(3) receptors uncovered a Ca(2+) response to LPA, which was manifest as a progressive increase in global cellular Ca(2+) with no discernible foci. We suggest that activation of an LPA-sensitive Edg-4 receptor solely utilizes the production of intracellular sphingosine 1-phosphate to stimulate Ca(2+) mobilization in SH-SY5Y cells. Unlike traditional Ca(2+) release processes, this novel pathway does not require the progressive recruitment of elementary Ca(2+) events.

Effect of dimethylsphingosine on muscarinic M(3) receptor signalling in SH-SY5Y cells.

The sphingosine kinase inhibitor, dimethylsphingosine, is an important tool for investigating intracellular effects of the putative second messenger compound, sphingosine 1-phosphate. However, the specificity of action of dimethylsphingosine has not been fully investigated. In human SH-SY5Y neuroblastoma cells, dimethylsphingosine (30 microM), produced a 25-fold increase in the EC(50) for methacholine-induced Ca(2+) mobilisation, and reduced the maximum response by 57+/-5%, suggesting the involvement of sphingosine 1-phosphate production in the Ca(2+) signal. However, dimethylsphingosine also inhibited [3H]N-methylscopolamine binding to whole SH-SY5Y cells and reduced methacholine-induced phosphoinositide turnover. Thus, this compound must be used with caution when investigating the role of sphingosine kinase in G-protein coupled receptor-mediated Ca(2+) mobilisation responses.

Complex involvement of pertussis toxin-sensitive G proteins in the regulation of type 1alpha metabotropic glutamate receptor signaling in baby hamster kidney cells.

Previously, we demonstrated that the coupling of the metabotropic glutamate receptor mGlu1alpha to phosphoinositide hydrolysis is enhanced by pertussis toxin (PTX) in stably transfected baby hamster kidney cells (BHK). Here, we show that the PTX effect on agonist-stimulated [(3)H]inositol phosphate accumulation can be resolved into two components: an immediate increase in agonist potency, and a more slowly developing increase in the magnitude of the response observed at maximally effective agonist concentrations. Using G(q/11)alpha- and G(i/o)alpha-selective antibodies to immunoprecipitate [(35)S]guanosine-5'-O-(3-thio)triphosphate-bound Galpha proteins, we also show that agonist stimulation of mGlu1alpha in BHK membranes increases specific [(35)S]guanosine-5'-O-(3-thio)triphosphate binding to both G(q/11) and G(i/o) proteins. Preincubation of BHK-mGlu1alpha with L-glutamate (300 microM) results in a progressive loss (60% in 30 min) of L-quisqualate-induced [(3)H]inositol phosphate accumulation (without a change in potency), providing evidence for agonist-induced receptor desensitization. Although such desensitization of mGlu receptor signaling was mimicked by a phorbol ester, agonist-induced phosphorylation of the receptor was not observed and protein kinase C inhibition by Ro 31-8220 did not prevent L-glutamate-mediated desensitization. In contrast, PTX treatment of the cells almost completely prevented L-glutamate-mediated desensitization. Together, these data provide evidence for a multifunctional coupling of mGlu1alpha to different types of G proteins, including PTX-sensitive G(i)-type G proteins. The latter are involved in the negative control of phospholipase C activity while also influencing the rate of desensitization of the mGlu1alpha receptor.

Intracellular Ca2+ stores regulate muscarinic receptor stimulation of phospholipase C in cerebellar granule cells.

Muscarinic receptor activation of phosphoinositide phospholipase C (PLC) has been examined in rat cerebellar granule cells under conditions that modify intracellular Ca2+ stores. Exposure of cells to medium devoid of Ca2+ for various times reduced carbachol stimulation of PLC with a substantial loss (88%) seen at 30 min. A progressive recovery of responses was observed following the reexposure of cells to Ca2+-containing medium (1.3 mM). However, these changes did not appear to result exclusively from changes in the cytosolic Ca2+ concentration ([Ca2+]i), which decreased to a lower steady level (approximately 25 nM decrease in 1-3 min after extracellular omission) and rapidly returned (within 1 min) to control values when extracellular Ca2+ was restored. Only after loading of the intracellular Ca2+ stores through a transient 1-min depolarization of cerebellar granule cells with 40 mM KCl, followed by washing in nondepolarizing buffer, was carbachol able to mobilize intracellular Ca2+. However, the same treatment resulted in an 80% enhancement of carbachol activation of PLC. In other experiments, partial depletion of the Ca2+ stores by pretreatment of cells with thapsigargin and caffeine resulted in an inhibition (18 and 52%, respectively) of the PLC response. Furthermore, chelation of cytosolic Ca2+ with BAPTA/AM did not influence muscarinic activation of PLC in either the control or predepolarized cells. These conditions, however, inhibited both the increase in [Ca2+]i and the PLC activation elicited by 40 mM KCl and abolished carbachol-induced intracellular Ca2+ release in predepolarized cells. Overall, these results suggest that muscarinic receptor activation of PLC in cerebellar granule cells can be modulated by changes in the loading state of the Ca2+ stores.

Receptor phosphorylation does not mediate cross talk between muscarinic M(3) and bradykinin B(2) receptors.

This study examined cross talk between phospholipase C-coupled muscarinic M(3) and bradykinin B(2) receptors coexpressed in Chinese hamster ovary (CHO) cells. Agonists of either receptor enhanced phosphoinositide signaling (which rapidly desensitized) and caused protein kinase C (PKC)-independent, homologous receptor phosphorylation. Muscarinic M(3) but not bradykinin B(2) receptors were also phosphorylated after phorbol ester activation of PKC. Consistent with this, muscarinic M(3) receptors were phosphorylated in a PKC-dependent fashion after bradykinin B(2) receptor activation, but muscarinic M(3) receptor activation did not influence bradykinin B(2) receptor phosphorylation. Despite heterologous phosphorylation of muscarinic M(3) receptors, phosphoinositide and Ca(2+) signaling were unaffected. In contrast, marked heterologous desensitization of bradykinin-mediated responses occurred despite no receptor phosphorylation. This desensitization was associated with a sustained component of muscarinic receptor-mediated signaling, whereas bradykinin's inability to influence muscarinic receptor-mediated responses was associated with rapid and full desensitization of bradykinin responses. Thus the mechanism of functional cross talk most likely involves depletion of a shared signaling component. These data demonstrate that receptor phosphorylation is not a prerequisite for heterologous desensitization and that such desensitization is not obligatory after heterologous receptor phosphorylation.

Lack of a C-terminal tail in the mammalian gonadotropin-releasing hormone receptor confers resistance to agonist-dependent phosphorylation and rapid desensitization.

The mammalian gonadotropin-releasing hormone receptor (GnRH-R) is, at present, the only G-protein-coupled receptor that activates phospholipase C and lacks a C-terminal tail. We have previously demonstrated that this unique structural feature is associated with resistance to rapid desensitization of phosphoinositide signaling in COS-7 and HEK-293 cells (Heding, A., Vrecl, M., Bogerd, J., McGregor, A., Sellar, R., Taylor, P. L., and Eidne, K. A. (1998) J. Biol. Chem. 273, 11472-11477). Using receptors tagged with a nonapeptide of the influenza hemagglutinin protein to enable immunoprecipitation, we now demonstrate that the mammalian GnRH-R is not phosphorylated in an agonist-dependent manner. In contrast, the mammalian thyrotropin-releasing hormone receptor and the African catfish GnRH-R, both of which have a C-terminal tail, are phosphorylated in response to agonist challenge. Furthermore, chimeras of the mammalian GnRH-R with the C-terminal tail of either the mammalian thyrotropin-releasing hormone receptor or the catfish GnRH-R are also phosphorylated in an agonist-dependent manner. Only those receptors having C-terminal tails showed desensitization of phosphoinositide responses within 5-10 min of agonist challenge. We also show that the internalization of all these receptors when expressed transiently in COS-7 cells is similar. This dissociates receptor internalization from rapid desensitization and demonstrates that the lack of a C-terminal tail in the mammalian GnRH-R results in an inability of the receptor to undergo agonist-dependent phosphorylation and that this results directly in a resistance to rapid desensitization.