Stimulation Of Phosphoinositide Hydrolysis Research Articles

Internalized β2-Adrenergic Receptors Oppose PLC-Dependent Hypertrophic Signaling.

Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to β-adrenergic receptor (β-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. β1-AR (β1-adrenergic receptor) and β2-ARs (β2-adrenergic receptor) are the 2 major subtypes of β-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of β1-ARs drives detrimental cardiac remodeling while β2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through β2-ARs remain unclear. β2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of β2-AR signaling mechanisms in the regulation of cardiac hypertrophy. Here, we show that β2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for β2-AR-mediated phospholipase C inhibition requires internalization of β2-AR, activation of Gi and Gβγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-β1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. This reveals a mechanism for β2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of β2-AR signaling on the development of heart failure.

Atypical signaling of metabotropic glutamate receptor 1 in human melanoma cells

The metabotropic glutamate 1 (mGlu1) receptor has emerged as a novel target for the treatment of metastatic melanoma and various other cancers. Our laboratory has demonstrated that a selective, non-competitive mGlu1 receptor antagonist slows human melanoma growth in vitro and in vivo. In this study, we sought to determine if the activation of a canonical G protein-dependent signal transduction cascade, which is often used as an output of mGlu1 receptor activity in neuronal cells, correlated with mGlu1 receptor-mediated melanoma cell viability. Glutamate, the endogenous ligand of mGlu1 receptors, significantly increased melanoma cell viability, but did not stimulate phosphoinositide (PI) hydrolysis in several human melanoma cell lines. In contrast, melanoma cell viability was not increased by quisqualate, a highly potent mGlu1 receptor agonist, or DHPG, a selective group I mGlu receptor agonist. Similarly to glutamate, quisqualate also failed to stimulate PI hydrolysis in mGlu1 receptor-expressing melanoma cells. These results suggest that the canonical G protein-dependent signal transduction cascade is not coupled to mGlu1 receptors in all human melanoma cells. On the other hand, dynamin inhibition selectively decreased viability of mGlu1 receptor-expressing melanoma cells, suggesting that a mechanism requiring internalization may control melanoma cell viability. Taken together, these data demonstrate that the approaches commonly used to study mGlu1 receptor function and signaling in other systems may be inappropriate for studying mGlu1 receptor-mediated melanoma cell viability.

Abstract A15: mGlu1 Receptors and downstream signal transduction proteins as therapeutic targets for the treatment of metastatic melanoma

Abstract The purpose of this study was to elucidate the downstream signaling of mGlu1 receptors involved in melanoma cell proliferation. Previously, using melanoma cell lines expressing mGlu1 receptors, we demonstrated that glutamate stimulation of mGlu1 receptors increases melanoma cell growth in BRAF mutant, NRAS mutant, and BRAF/NRAS wild-type melanoma cells. Blockade of mGlu1 receptor activation through glutamate depletion, mGlu1 receptor knockdown, and mGlu1 receptor antagonism inhibited glutamate-stimulated melanoma cell growth, further identifying mGlu1 receptors as a therapeutic target for the treatment of metastatic melanoma. Because mGlu1 receptor antagonists blocked growth of both BRAF and NRAS mutant melanoma cells, it suggests that mGlu1 receptors stimulate melanoma cell proliferation either by a mechanism downstream of NRAS and BRAF or through a distinct and necessary pathway. Here we report that glutamate does not stimulate melanoma cell proliferation through activation of the canonical G alpha-q dependent pathway, as neither glutamate nor quisqualate stimulate phosphoinositide hydrolysis in the mGlu1 receptor-expressing melanoma cell lines SK-MEL-2 and SK-MEL-5 (as measured by scintillation proximity assay). Furthermore, the G protein biased ligand quisqualate does not stimulate SK-MEL-2 or SK-MEL-5 melanoma cell growth (as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay). In contrast, MEK inhibitors block glutamate stimulated growth of SK-MEL-2 and SK-MEL-5 melanoma cells, indicating that the MEK/ERK signal transduction cascade is involved in glutamate dependent growth. Our laboratory has shown in stably transfected CHO cells that mGlu1a receptors can also activate a G protein independent, Beta-arrestin-1 dependent pathway, and this Beta-arrestin-1 dependent signaling requires internalization of mGlu1 receptors. To assess the involvement of Beta-arrestin-1 dependent signaling in glutamate stimulated melanoma growth, cells were treated with a dynamin inhibitor, thus preventing internalization. Inhibition of dynamin selectively blocked glutamate stimulated growth of mGlu1 receptor expressing melanomas, but did not affect proliferation of melanoma cells lacking mGlu1 receptor expression. In summary, these data suggest that glutamate stimulates melanoma cell growth through activation of a dynamin-dependent pathway which may result in ERK phosphorylation. Understanding the signaling mechanism downstream of mGlu1 receptors may lead to the identification of novel targets that could be used either separately or in combination with mGlu1 receptor antagonists for the treatment of metastatic melanoma. NIH Grant NS37436 PhRMA Pre Doctoral Fellowship in Pharmacology/Toxicology Citation Format: Tara Gelb, Sergey Pshenichkin, Hannah A. Hathaway, Ewa Grajkowska, Barry B. Wolfe, Jarda T. Wroblewski. mGlu1 Receptors and downstream signal transduction proteins as therapeutic targets for the treatment of metastatic melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Melanoma: From Biology to Therapy; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(14 Suppl):Abstract nr A15.

The C2 Domain and Altered ATP-Binding Loop Phosphorylation at Ser³⁵⁹ Mediate the Redox-Dependent Increase in Protein Kinase C-δ Activity.

The diverse roles of protein kinase C-δ (PKCδ) in cellular growth, survival, and injury have been attributed to stimulus-specific differences in PKCδ signaling responses. PKCδ exerts membrane-delimited actions in cells activated by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Tyr(313)-phosphorylated enzyme that displays a high level of lipid-independent activity and altered substrate specificity during oxidative stress. This study identifies an interaction between PKCδ's Tyr(313)-phosphorylated hinge region and its phosphotyrosine-binding C2 domain that controls PKCδ's enzymology indirectly by decreasing phosphorylation in the kinase domain ATP-positioning loop at Ser(359). We show that wild-type (WT) PKCδ displays a strong preference for substrates with serine as the phosphoacceptor residue at the active site when it harbors phosphomimetic or bulky substitutions at Ser(359.) In contrast, PKCδ-S359A displays lipid-independent activity toward substrates with either a serine or threonine as the phosphoacceptor residue. Additional studies in cardiomyocytes show that oxidative stress decreases Ser(359) phosphorylation on native PKCδ and that PKCδ-S359A overexpression increases basal levels of phosphorylation on substrates with both phosphoacceptor site serine and threonine residues. Collectively, these studies identify a C2 domain-pTyr(313) docking interaction that controls ATP-positioning loop phosphorylation as a novel, dynamically regulated, and physiologically relevant structural determinant of PKCδ catalytic activity.

Mechanisms that regulate PKCδ‐dependent phosphorylation of cardiac troponin I: the role of the C2 domain and ATP‐binding loop phosphorylation S357 (1081.2)

PKCδ's diverse roles in cell growth, survival, and injury have been attributed to stimulus‐specific differences in PKCδ‐dependent signaling responses. PKCδ exerts membrane‐delimited actions following activation by agonists that stimulate phosphoinositide hydrolysis. PKCδ is released from membranes as a Y311‐phosphorylated enzyme that displays a high level of lipid‐independent activity and altered substrate specificity during oxidative stress. We previously showed that PKCδ phosphorylates cardiac troponin I (cTnI) at S23/S24 when it is allosterically‐activated by lipid‐cofactors and that PKCδ acquires activity toward an additional site on cTnI (T144) during oxidative stress when it is Y311‐phosphorylated by Src. This study identifies an interaction between PKCδ’s phosphotyrosine‐binding C2 domain and the Y311‐phosphorylated hinge region (which resides in a C2 domain consensus‐binding motif) that controls PKCδ's enzymology indirectly by regulating ATP‐positioning loop phosphorylation at S357. Mutagenesis studies show that PKCδ‐S357A is a lipid‐independent enzyme that displays high levels of cTnI‐S23/S24 and T141 kinase activity. In contrast, phosphomimetic or bulky substitutions at S357 prevent cTnI phosphorylation at T141 (but not S23/S24) due to an effect on the enzyme’s phosphoacceptor site selectivity. S‐A substitutions at cognate sites in the highly‐conserved ATP‐binding loops of PKCα and PKA increase catalytic activity, but do not alter phosphoacceptor site selectivity. The C2 domain‐pY311 docking interaction that controls ATP‐binding loop phosphorylation represents a novel structural determinant of PKCδ catalytic activity.Grant Funding Source: Supported by HL77860

Novel Structural and Functional Insights into M3 Muscarinic Receptor Dimer/Oligomer Formation

Class A G protein-coupled receptors (GPCRs) are able to form homodimers and/or oligomeric arrays. We recently proposed, based on bioluminescence resonance energy transfer studies with the M3 muscarinic receptor (M3R), a prototypic class A GPCR, that the M3R is able to form multiple, structurally distinct dimers that are probably transient in nature (McMillin, S. M., Heusel, M., Liu, T., Costanzi, S., and Wess, J. (2011) J. Biol. Chem. 286, 28584-28598). To provide more direct experimental support for this concept, we employed a disulfide cross-linking strategy to trap various M3R dimeric species present in a native lipid environment (transfected COS-7 cells). Disulfide cross-linking studies were carried out with many mutant M3Rs containing single cysteine (Cys) substitutions within two distinct cytoplasmic M3R regions, the C-terminal portion of the second intracellular loop (i2) and helix H8 (H8). The pattern of cross-links that we obtained, in combination with molecular modeling studies, was consistent with the existence of two structurally distinct M3R dimer interfaces, one involving i2/i2 contacts (TM4-TM5-i2 interface) and the other one characterized by H8-H8 interactions (TM1-TM2-H8 interface). Specific H8-H8 disulfide cross-links led to significant impairments in M3R-mediated G protein activation, suggesting that changes in the structural orientation or mobility of H8 are critical for efficient receptor-G protein coupling. Our findings provide novel structural and functional insights into the mechanisms involved in M3R dimerization (oligomerization). Because the M3R shows a high degree of sequence similarity with many other class A GPCRs, our findings should be of considerable general interest.

Characterization of signaling pathways coupled to melatonin receptors in gastrointestinal smooth muscle

Melatonin, a close derivative of serotonin, is involved in physiological regulation of circadian rhythms. In the gastrointestinal (GI) system, melatonin exhibits endocrine, paracrine and autocrine actions and is implicated in the regulation of GI motility. However, it is not known whether melatonin can also act directly on GI smooth muscle cells. The aim of the present study was to determine the expression of melatonin receptors in smooth muscle and identify their signaling pathways. MT1, but not MT2 receptors are expressed in freshly dispersed and cultured gastric smooth muscle cells. Melatonin selectively activated Gq and stimulated phosphoinositide (PI) hydrolysis in freshly dispersed and cultured muscle cells. PI hydrolysis was blocked by the expression of Gq, but not Gi minigene in cultured muscle cells. Melatonin also caused rapid increase in cytosolic Ca2+ as determined by epifluorescence microscopy in fura-2 loaded single smooth muscle cells, and induced rapid contraction. Melatonin-induced PI hydrolysis and contraction were blocked by a non-selective MT1/MT2 antagonist luzindole (1μM), but not by a selective MT2 antagonist 4P-PDOT (100nM), and by the PLC inhibitor U73122. MT2 selective agonist IIK7 (100nM) had no effect on PI hydrolysis and contraction. We conclude that rabbit gastric smooth muscle cells express melatonin MT1 receptors coupled to Gq. Activation of these receptors causes stimulation of PI hydrolysis and increase in cytosolic Ca2+, and elicits muscle contraction.

A basic residue in the proximal C-terminus is necessary for efficient activation of the M-channel subunit Kv7.2 by PI(4,5)P2

All Kv7 potassium channels require membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) for their normal function and hence can be physiologically regulated by neurotransmitters and hormones that stimulate phosphoinositide hydrolysis. Recent mutational analysis indicates that a cluster of basic residues in the proximal C-terminus (K354/K358/R360/K362) is crucial for PI(4,5)P2 activation of cardiac Kv7.1 channels. Since this cluster is largely conserved in all Kv7 subunits, we tested whether homologous residues are also required for activation of Kv7.2 (a subunit of neuronal M-channels). We found that the mutation Kv7.2 (R325A) (corresponding to R360 in Kv7.1) reduced Kv7.2 current amplitude by ∼60 % (P < 0.02) without change in voltage sensitivity and reduced the sensitivity of Kv7.2 channels to dioctanoyl-phosphatidylinositol-4,5-bisphosphate by ∼eightfold (P < 0.001). Taking into account previous experiments (Zhang et al., Neuron 37:963–75, 2003) implicating Kv7.2 (H328), and since R325 and H328 are conserved in homologous positions in all other Kv7 channels, we suggest that this proximal C-terminal domain adjacent to the last transmembrane domain that contains R325 and H328 (in Kv7.2) might play a major role in the activation of all members of the Kv7 channel family by PI(4,5)P2.

Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic β cells

Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic β cells. In the present study, we have identified the expression of TGR5 in pancreatic β cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated Gαs and caused an increase in intracellular cAMP and Ca2+. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective Gαs inhibitor) or U73122 (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, U73122 or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on Gs/cAMP/Ca2+ pathway. 8-pCPT-2′-O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic β cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

Thrombin Receptor Regulates Hematopoiesis and Endothelial-to-Hematopoietic Transition

Hematopoietic development and vascular development are closely related physiological processes during vertebrate embryogenesis. Recently, endothelial-to-hematopoietic transition (EHT) was demonstrated to be critical for hematopoietic stem and progenitor cell induction, but its underlying regulatory mechanisms remain poorly understood. Here we show that thrombin receptor (F2r), a protease-activated G protein-coupled receptor required for vascular development, functions as a negative regulator during hematopoietic development. F2r is significantly upregulated during hematopoietic differentiation of mouse embryonic stem cells (mESCs) and zebrafish hematopoietic development. Pharmacological or genetic inhibition of F2r promotes hematopoietic differentiation, whereas F2r overexpression shows opposite effects. Further mechanistic studies reveal that F2r-RhoA/ROCK pathway inhibits EHT in vitro and negatively regulates zebrafish EHT and hematopoietic stem cell induction in vivo. Taken together, this study demonstrates a fundamental role of F2r-RhoA/ROCK pathway in vertebrate hematopoiesis and EHT, as well as an important molecular mechanism coordinating hematopoietic and vascular development.

Kinin B2 Receptor‐Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15‐Dependent Intracellular Bradykinin Degradation

Kinins are potent proinflammatory peptides that are produced extracellularly and rapidly degraded by extracellular and intracellular peptidases. In this study, we developed model cell systems expressing the kinin B2 receptor (B2R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B2R. No direct colocalization of B2R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B2R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular calcium mobilization. Cell surface‐bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15‐dependent manner upon B2R‐mediated endocytosis. These results show that EP24.15 acts to negatively regulate B2R responsiveness and as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.

Selectivity of Agonists for the Active State of M1 to M4 Muscarinic Receptor Subtypes

We measured the intrinsic relative activity (RA(i)) of muscarinic agonists to detect possible selectivity for receptor subtypes and signaling pathways. RA(i) is a relative measure of the microscopic affinity constant of an agonist for the active state of a GPCR expressed relative to that of a standard agonist. First, we estimated RA(i) values for a panel of agonists acting at the M(4) muscarinic receptor coupled to three distinct G-protein pathways: G(i) inhibition of cAMP accumulation, G(s) stimulation of cAMP accumulation, and G alpha(15) stimulation of phosphoinositide hydrolysis. Our results show similar RA(i) values for each agonist, suggesting that the same active state of the M(4) receptor triggers the activation of the three G proteins. We also estimated RA(i) values for agonists across M(1) to M(4) muscarinic subtypes stably transfected in Chinese hamster ovary cells. Our results show selectivity of McN-A-343 [4-I-[3-chlorophenyl]carbamoyloxy)-2-butynyltrimethylammnonium chloride] for the M(1) and M(4) subtypes and selectivity of pilocarpine for the M(1) and M(3) subtypes. The other agonists tested lacked marked selectivity among M(1) to M(4) receptors. Finally, we estimated RA(i) values from published literature on M(1), M(2), and M(3) muscarinic responses and obtained results consistent with our own studies. Our results show that the RA(i) estimate is a useful receptor-dependent measure of agonist activity.

Kinin B2Receptor-Mediated Bradykinin Internalization and Metalloendopeptidase EP24.15-Dependent Intracellular Bradykinin Degradation

Kinins are potent proinflammatory peptides that are produced extracellularly and are rapidly degraded by extracellular peptidases and by intracellular peptidases accessed by kinins via receptor-mediated endocytosis. In this study, we developed model cell systems expressing the kinin B(2) receptor (B(2)R) and the metalloendopeptidase thimet oligopeptidase (EC 3.4.24.15; EP24.15) either individually or together to address 1) the cellular and functional relationship between these proteins and 2) the participation of EP24.15 in the metabolism of bradykinin (BK) after BK internalization via B(2)R. B(2)R was localized almost exclusively in the plasma membrane, whereas EP24.15 was localized both intracellularly and on the cell surface and secreted in the media. Intracellular EP24.15 was present throughout the cell, both cytosolic and particulate, with less nuclear localization and no colocalization with either the endoplasmic reticulum marker calnexin or the Golgi marker GM130. No direct colocalization of B(2)R and EP24.15 was observed using immunofluorescence microscopy. However, the two proteins coimmunoprecipitated specifically, and EP24.15 attenuated maximal B(2)R responsiveness without influencing the potency of BK to stimulate phosphoinositide hydrolysis and intracellular Ca(2+) mobilization. Cell surface-bound BK remained intact in cells overexpressing EP24.15 but was degraded intracellularly in an EP24.15-dependent manner upon B(2)R-mediated endocytosis. These results show that EP24.15 acts to negatively regulate B(2)R responsiveness, and it serves as an intracellular peptidase in the degradation of BK specifically internalized via this receptor.

UDP Is a Competitive Antagonist at the Human P2Y14Receptor

G protein-coupled P2Y receptors (P2Y-R) are activated by adenine and uracil nucleotides. The P2Y(14) receptor (P2Y(14)-R) is activated by at least four naturally occurring UDP sugars, with UDP-glucose (UDP-Glc) being the most potent agonist. With the goal of identifying a competitive antagonist for the P2Y(14)-R, UDP was examined for antagonist activity in COS-7 cells transiently expressing the human P2Y(14)-R and a chimeric Galpha protein that couples Gi-coupled receptors to stimulation of phosphoinositide hydrolysis. UDP antagonized the agonist action of UDP-Glc, and Schild analysis confirmed that the antagonism was competitive (pK(B) = 7.28). Uridine 5'-O-thiodiphosphate also antagonized the human P2Y(14)-R (hP2Y(14)-R) with an apparent affinity similar to that of UDP. In contrast, no antagonist activity was observed with ADP, CDP, or GDP, and other uracil analogs also failed to exhibit antagonist activity. The antagonist activity of UDP was not observed at other hP2Y-R. In contrast to its antagonist action at the hP2Y(14)-R, UDP was a potent agonist (EC(50) = 0.35 muM) at the rat P2Y(14)-R. These results identify the first competitive antagonist of the P2Y(14)-R and demonstrate pharmacological differences between receptor orthologs.

Functional Effects of Monoclonal Antibodies to the Purified Amino-Terminal Extracellular Domain of the Human Ca2+ Receptor

We generated three functionally unique monoclonal antibodies to the purified human CaR extracellular domain. Flow cytometry studies of chimeric receptors localized their epitopes to lobe 2 of the VFT domain. These results lead us to propose a mechanism for the functional effects of these antibodies. The human Ca(2+) receptor (CaR), which plays a central role in the regulation of [Ca(2+)](0) homeostasis, has a distinctively large extracellular domain that consists of a bilobed Venus flytrap (VFT) domain, involved in agonist binding, and a cysteine-rich domain. Functional antibodies that specifically bind to this domain would have therapeutic potential and could be used as a tool to gain insights into receptor activation as well. We generated three monoclonal antibodies (mAbs), 7F8, 5C8, and 1A8, to the purified human CaR extracellular domain. Functional characterization of these antibodies included Ca(2+) stimulation of phosphoinositide hydrolysis to examine effects of intact or protease digested antibodies on sensitivity of the receptor to extracellular Ca(2+) and flow cytometry assay of binding of the antibodies to HEK-293 cells expressing chimeric receptors to map antibody epitopes. We found these mAbs specifically recognize native but not denatured human CaR or homologous native Fugu CaR. Sensitivity of the human CaR to extracellular calcium was increased by binding of 5C8 but decreased by binding of 1A8. A chimeric receptor FCFCF, with lobe 2 region of the human CaR VFT domain in the Fugu CaR backbone, bound all three mAbs, and the sensitivity of this chimeric CaR to extracellular Ca(2+) was also increased by binding of 5C8 and decreased by binding of 1A8. The epitopes of these mAbs reside in the lobe 2 region of the human CaR VFT domain. 5C8 might activate the receptor by facilitating closure and/or rotation of the VFT domains on agonist binding, whereas 1A8 might inhibit the receptor by impeding such agonist-induced conformational changes. Recombinant antibodies with antigen binding domains of 5C8 and 1A8 could be useful in the treatment of hyperparathyroidism and osteoporosis, respectively.

Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M2 Muscarinic Receptor Signaling through Gi/o,Gs, and G15

We developed novel methods for analyzing the concentration-response curve of an agonist to estimate the product of observed affinity and intrinsic efficacy, expressed relative to that of a standard agonist. This parameter, termed intrinsic relative activity (RA(i)), is most applicable for the analysis of responses at G protein-coupled receptors. RA(i) is equivalent to the potency ratios that agonists would exhibit in a hypothetical, highly sensitive assay in which all agonists behave as full agonists, even those with little intrinsic efficacy. We investigated muscarinic responses at the M(2) receptor, including stimulation of phosphoinositide hydrolysis through G(alpha15) in HEK 293T cells, inhibition of cAMP accumulation through G(i) in Chinese hamster ovary (CHO) cells, and stimulation of cAMP accumulation through G(s) in CHO cells treated with pertussis toxin. The RA(i) values of carbachol, oxotremorine-M, and the enantiomers of aceclidine were approximately the same in the three assay systems. In contrast, the activity of 4-[[N-[3-chlorophenyl]carbamoy]oxy-2-butynyl]trimethylammonium chloride (McN-A-343) was approximately 10-fold greater at M(2) receptors coupled to G(alpha15) in HEK 293T cells compared with M(2) receptors coupled to G(i) in the same cells or in CHO cells. Our results show that the RA(i) estimate is a useful measure for quantifying agonist activity across different assay systems and for detecting agonist directed signaling.

The Spatial Distribution of Inositol 1,4,5-Trisphosphate Receptor Isoforms Shapes Ca2+ Waves

Cytosolic Ca(2+) is a versatile second messenger that can regulate multiple cellular processes simultaneously. This is accomplished in part through Ca(2+) waves and other spatial patterns of Ca(2+) signals. To investigate the mechanism responsible for the formation of Ca(2+) waves, we examined the role of inositol 1,4,5-trisphosphate receptor (InsP3R) isoforms in Ca(2+) wave formation. Ca(2+) signals were examined in hepatocytes, which express the type I and II InsP3R in a polarized fashion, and in AR4-2J cells, a nonpolarized cell line that expresses type I and II InsP3R in a ratio similar to what is found in hepatocytes but homogeneously throughout the cell. Expression of type I or II InsP3R was selectively suppressed by isoform-specific DNA antisense in an adenoviral delivery system, which was delivered to AR4-2J cells in culture and to hepatocytes in vivo. Loss of either isoform inhibited Ca(2+) signals to a similar extent in AR4-2J cells. In contrast, loss of the basolateral type I InsP3R decreased the sensitivity of hepatocytes to vasopressin but had little effect on the initiation or spread of Ca(2+) waves across hepatocytes. Loss of the apical type II isoform caused an even greater decrease in the sensitivity of hepatocytes to vasopressin and resulted in Ca(2+) waves that were much slower and delayed in onset. These findings provide evidence that the apical concentration of type II InsP3Rs is essential for the formation of Ca(2+) waves in hepatocytes. The subcellular distribution of InsP3R isoforms may critically determine the repertoire of spatial patterns of Ca(2+) signals.

Topographical resolution of jaw movements mediated by cyclase- vs. non-cyclase-coupled dopamine D 1-like receptors: Studies with SK&F 83822

This study examined the effects on orofacial movement topography of SK&F 83822 ([ R/ S]-6-chloro-7,8-dihydroxy-3-allyl-1-[3-methylphenyl]-2,3,4,5-tetrahydro-1 H-3-benzazepine), which stimulates dopamine D 1-like receptors coupled to stimulation of adenylyl cyclase (AC) but not phosphoinositide (PI) hydrolysis, in comparison with SK&F 83959 ([ R/ S]-3-methyl-6-chloro-7,8-dihydroxy-1-[3-methyl-phenyl]-2,3,4,5-tetrahydro-1 H-3-benzazepine), which stimulates PI hydrolysis but not AC. SK&F 83822 alone induced chattering, while SK&F 83959 alone exerted little effect. SK&F 83822 and SK&F 83959 each in combination with the dopamine D 2-like agonist quinpirole resulted in synergistic induction of non-chattering movements with tongue protrusions. These effects were blocked by the dopamine D 1-like receptor antagonist SCH 23390 ([ R]-3-methyl-7-chloro-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1 H-3-benzazepine). However, the dopamine D 2-like receptor antagonist YM 09151-2 ( cis- N-[1-benzyl-2-methyl-pyrrolidin-3-yl]-5-chloro-2-methoxy-4-methylaminobenzamide) exerted a biphasic effect on synergism with SK&F 83822: chattering was initially released but antagonised thereafter. Only antagonism was seen for synergism with SK&F 83959. While both AC- and PI-coupled dopamine D 1-like receptors participate in synergistic dopamine D 1-like:D 2-like receptor interactions, topographically specific synergistic and oppositional dopamine D 1-like:D 2-like interactions evident with SK&F 83822 reflect the involvement primarily of D 1-like receptors coupled to AC rather than PI.

Granulocyte function is stimulated by a novel hexapeptide, WKYMVm, in chemotherapy-treated cancer patients

Bacterial infections are major life-threatening complications in patients receiving cytotoxic drugs. These infections generally occur during periods of neutropenia. It has been suggested that the incidence of neutropenia correlates with the incidence of infections. A synthetic hexapeptide, WKYMVm, which stimulates phosphoinositide hydrolysis in leukocytes, has been shown to activate microbicidal activities of human polymorphonuclear neutrophils. In this study, we evaluate whether WKYMVm stimulates bactericidal activity in neutrophils obtained from patients who received chemotherapy for solid tumors when they were neutropenic. Eight patients and 11 healthy controls were recruited for the study. Patient neutrophils, on day 0 and at 2 weeks after chemotherapy, were collected. Expression of the WKYMVm peptide receptor, on leukocytes, was analyzed by fluorescein-activated cell sorting. Neutrophil bactericidal assays were performed using both reactive oxygen species generation and intracellular killing. Expression of the WKYMVm peptide receptor on leukocytes showed no difference in the treated patients compared to healthy controls. WKYMVm increased bactericidal activities, in a dose-dependent fashion, of control neutrophils compared to treated patient neutrophils obtained on day 0. WKYMVm markedly stimulated bactericidal activity of treated patient neutrophils obtained at 2 weeks after chemotherapy compared to treated patient neutrophils obtained on day 0. WKYMVm augmented neutrophil bactericidal activity was noted at low concentration but was suppressed at higher concentrations of 5-fluorouracil. WKYMVm augmented neutrophil bactericidal activity was not suppressed by cisplatin. WKYMVm has the potential for increasing neutrophil bactericidal activity in chemotherapy-treated cancer patients.

A requirement for membrane cholesterol in the β-arrestin- and clathrin-dependent endocytosis of LPA1 lysophosphatidic acid receptors

Lysophosphatidic acid (LPA) stimulates heterotrimeric G protein signaling by activating three closely related receptors, termed LPA(1), LPA(2) and LPA(3). Here we show that in addition to promoting LPA(1) signaling, membrane cholesterol is essential for the association of LPA(1) with beta-arrestin, which leads to signal attenuation and clathrin-dependent endocytosis of LPA(1). Reduction of clathrin heavy chain expression, using small interfering RNAs, inhibited LPA(1) endocytosis. LPA(1) endocytosis was also inhibited in beta-arrestin 1 and 2-null mouse embryo fibroblasts (beta-arrestin 1/2 KO MEFs), but was restored upon re-expression of wild-type beta-arrestin 2. beta-arrestin attenuates LPA signaling as LPA(1)-dependent phosphoinositide hydrolysis was significantly elevated in beta-arrestin 1/2 KO MEFs and was reduced to wild-type levels upon re-expression of wild-type beta-arrestin. Interestingly, extraction of membrane cholesterol with methyl-beta-cyclodextrin inhibited LPA(1) signaling, beta-arrestin membrane recruitment and LPA(1) endocytosis. Cholesterol repletion restored all of these functions. However, neither the stimulation of phosphoinositide hydrolysis by the M(1) acetylcholine receptor nor its endocytosis was affected by cholesterol extraction. LPA treatment increased the detergent resistance of LPA(1) and this was inhibited by cholesterol extraction, suggesting that LPA(1) localizes to detergent-resistant membranes upon ligand stimulation. These data indicate that although LPA(1) is internalized by clathrin- and beta-arrestin dependent endocytosis, membrane cholesterol is critical for LPA(1) signaling, membrane recruitment of beta-arrestins and LPA(1) endocytosis.