Number Of Cell Surface Receptors Research Articles

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases.

Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild-type (wt) and also a gain-of-function mutant of Btk are redistributed following a wide range of receptor-mediated stimuli through phosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3-K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-function mutant of Btk was found to be a potent inducer of lamellipodia and/or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3-K and certain members of the Rho-family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

Redistribution of Bruton's tyrosine kinase by activation of phosphatidylinositol 3-kinase and Rho-family GTPases

Bruton's tyrosine kinase (Btk) is a member of the Tec family of protein tyrosine kinases (PTK) characterized by an N-terminal pleckstrin homology domain (PH) thought to directly interact with phosphoinositides. We report here that wild-type (wt) and also a gain-of-function mutant of Btk are redistributed following a wide range of receptor-mediated stimuli through phosphatidylinositol 3-kinase (PI 3-K) activation. Employing chimeric Btk with green fluorescent protein in transient transfections resulted in Btk translocation to the cytoplasmic membrane of live cells through various forms of upstream PI 3-K activation. The redistribution was blocked by pharmacological and biological inhibitors of PI 3-K. A gain-of-function mutant of Btk was found to be a potent inducer of lamellipodia and / or membrane ruffle formation. In the presence of constitutively active forms of Rac1 and Cdc42, Btk is co-localized with actin in these regions. Formation of the membrane structures was blocked by the dominant negative form of N17-Rac1. Therefore, Btk forms a link between a vast number of cell surface receptors activating PI 3-K and certain members of the Rho-family of small GTPases. In the chicken B cell line, DT40, cells lacking Btk differed from wt cells in the actin pattern and showed decreased capacity to form aggregates, further suggesting that cytoskeletal regulation mediated by Btk may be of physiological relevance.

Granulocyte macrophage colony-stimulating factor and interleukin-3 regulate chemokine and chemokine receptor expression in bone marrow macrophages

The β-chemokine macrophage inflammatory protein-1 alpha (MIP-1α) and its associated receptors are involved in the regulation of pro-inflammatory and haemopoietic processes. This study was designed to investigate regulation of expression MIP-1α and its receptors by other haemopoietic cytokines. Murine bone marrow macrophages (BMM) were treated with or without GM-CSF or IL-3 and expression of MIP-1α, other chemokines and their receptors examined by Northern blotting. Receptor levels were also examined using Scatchard analysis and functional tests. Treatment of BMM with GM-CSF revealed a striking increase in MIP-1α mRNA levels, relative to untreated cells with a corresponding increase in MIP-1α protein. A similar increase in mRNA levels was found when BMM were treated with IL-3. An increase in the expression of three other β-chemokines namely MIP-1β, MCP-1 and MCP-3, was also found following treatment with GM-CSF or IL-3. We have additionally examined the expression of the known β-chemokine receptors in BMM and observed an increase in CCR1 mRNA levels following treatment with GM-CSF and IL-3, but no change was seen in the level of CCR5 expression. The increase in CCR1 expression was reflected in an increase in the number of cell surface receptors for MIP-1α on the GM-CSF treated BMM and in an enhanced response of the GM-CSF treated BMM to CCR1 ligands. These data suggest that GM-CSF and IL-3 may be involved in mechanisms regulating expression levels of MIP-1α and its receptors.

Cell Surface Stability of γ-Aminobutyric Acid Type A Receptors: DEPENDENCE ON PROTEIN KINASE C ACTIVITY AND SUBUNIT COMPOSITION

Type A gamma-aminobutyric acid receptors (GABA(A)), the major sites of fast synaptic inhibition in the brain, are believed to be composed predominantly of alpha, beta, and gamma subunits. Although cell surface expression is essential for GABA(A) receptor function, little is known regarding its regulation. To address this issue, the membrane stability of recombinant alpha(1)beta(2) or alpha(1)beta(2)gamma(2) receptors was analyzed in human embryonic kidney cells. Alpha(1)beta(2)gamma(2) but not alpha(1)beta(2) receptors were found to recycle constitutively between the cell surface and a microtubule-dependent, perinuclear endosomal compartment. Similar GABA(A) receptor endocytosis was also seen in cultured hippocampal and cortical neurons. GABA(A) receptor surface levels were reduced upon protein kinase C (PKC) activation. Like basal endocytosis, this response required the gamma(2) subunit but not receptor phosphorylation. Although inhibiting PKC activity did not block alpha(1)beta(2)gamma(2) receptor endocytosis, it did prevent receptor down-regulation, suggesting that PKC activity may block alpha(1)beta(2)gamma(2) receptor recycling to the cell surface. In agreement with this observation, blocking recycling from endosomes with wortmannin selectively reduced surface levels of gamma(2)-containing receptors. Together, our results demonstrate that the surface stability of GABA(A) receptors can be dynamically and specifically regulated, enabling neurons to modulate cell surface receptor number upon the appropriate cues.

Divergent Signaling Pathways Link Focal Adhesion Kinase to Mitogen-activated Protein Kinase Cascades: EVIDENCE FOR A ROLE OF PAXILLIN IN c-Jun NH2-TERMINAL KINASE ACTIVATION

Stimulation of a number of cell surface receptors, including integrins and G protein-coupled receptors, results in the activation of a non-receptor tyrosine kinase known as focal adhesion kinase (FAK). In turn, this kinase is believed to play a critical role in signaling to intracellular kinase cascades controlling gene expression such as extracellular signal-regulated kinases (ERKs), by a yet poorly defined mechanism. Furthermore, whether this tyrosine kinase also mediates the activation of other mitogen-activated protein kinase family members, such as c-Jun NH(2)-terminal kinases (JNKs), is still unclear. We show here that the activation of FAK by anchoring to the cell membrane is itself sufficient to stimulate potently both ERK and JNK. These effects were found to be phosphatidylinositol 3-kinase-independent, as FAK effectively stimulated Akt, and wortmannin suppressed Akt but not ERK or JNK activation. As previously reported by others, activation of ERK correlated with the ability of FAK to induce tyrosine phosphorylation of Shc. Surprisingly, however, stimulation of JNK was not dependent on the kinase activity of FAK or on the ability to induce tyrosine phosphorylation of FAK substrates. Instead, we provide evidence that FAK may stimulate JNK through a novel pathway involving the recruitment of paxillin to the plasma membrane and the subsequent activation of a biochemical route dependent on small GTP-binding proteins of the Rho family.

Regulation of insulin-like growth factor I receptor promoter activity by wild-type and mutant versions of the WT1 tumor suppressor.

The insulin-like growth factor I (IGF-I) receptor is a transmembrane tyrosine kinase that mediates the growth-promoting effects of IGF-I and IGF-II. Changes in IGF-I receptor messenger RNA levels are reflected in cell surface receptor number, and modulation of IGF-I receptor levels affects tumorigenicity in numerous cellular models; thus, control of IGF-I receptor gene expression appears to be an important level at which cellular proliferation and tumorigenic potential may be regulated. We have previously shown that the product of the WT1 Wilms' tumor suppressor gene represses IGF-I receptor gene expression both in vitro and in vivo, and that decreased WT1 levels are correlated with up-regulation of IGF-I receptor gene expression in Wilms' tumor, benign prostatic hyperplasia, and breast cancer. Gene regulation by WT1 is complex, in that the WT1 gene encodes a variety of products as a result of alternative splicing and RNA editing, and a number of missense point mutations have been characterized in Wilms' tumor-associated syndromes. Additionally, the WT1 protein has been demonstrated to self-associate through its N-terminal domain, although the role of this intermolecular interaction in transcriptional regulation by WT1 is unclear. In this report, we analyze the relative activity of wild-type and mutant versions of the WT1 protein with respect to IGF-I receptor promoter activity in transient transfection assays and assess the potential contribution of WT1 self-association to IGF-I receptor regulation using the yeast two-hybrid system. Of the naturally occurring variations in WT1 structure, only the presence of a three-amino acid KTS insert in the zinc finger domain introduced by alternative splicing of exon 9 had a significant effect on WT1 repression of IGF-I receptor promoter activity. The N- and C-terminal domains of WT1 also exhibited partial repression, as did the most common mutant version of the WT1 protein associated with Denys-Drash syndrome. Mutations in the WT1 N-terminus attenuated WT1 self-association in the yeast two-hybrid system, but did not impair transcriptional repression. Our results suggest that 1) the DNA-binding capacity of WT1 is critical for maximal repression of the IGF-I receptor promoter, but some effects may be mediated through protein-protein interactions involving the N-terminal domain; 2) WT1 self-association may not be required for repression of the IGF-I receptor promoter; and 3) the Denys-Drash syndrome version of the WT1 protein may exhibit residual or possible gain of function activity in some contexts rather than exerting dominant negative effects, as has been proposed previously.

The C-terminus of the kinase-defective neuregulin receptor ErbB-3 confers mitogenic superiority and dictates endocytic routing.

Signaling by the epidermal growth factor (EGF) family and the neuregulin group of ligands is mediated by four ErbB receptor tyrosine kinases, that form homo- and heterodimeric complexes. Paradoxically, the neuregulin receptor ErbB-3 is devoid of catalytic activity, but its heterodimerization with other ErbBs, particularly the ligand-less ErbB-2 oncoprotein of carcinomas, reconstitutes superior mitogenic and transforming activities. To understand the underlying mechanism we constructed a chimeric EGF-receptor (ErbB-1) whose autophosphorylation C-terminal domain was replaced by the corresponding portion of ErbB-3. Consistent with the possibility that this domain recruits a relatively potent signaling pathway(s), the mitogenic signals generated by the recombinant fusion protein were superior to those generated by ErbB-1 homodimers and comparable to the proliferative activity of ErbB-2/ErbB-3 heterodimers. Upon ligand binding, the chimeric receptor recruited an ErbB-3-specific repertoire of signaling proteins, including Shc and the phosphatidylinositol 3-kinase, but excluding the ErbB-1-specific substrate, phospholipase Cgamma1. Unlike ErbB-1, which is destined to lysosomal degradation through a mechanism that includes recruitment of c-Cbl and receptor poly-ubiquitination, the C-terminal tail of ErbB-3 shunted the chimeric protein to the ErbB-3-characteristic recycling pathway. These observations attribute the mitogenic superiority of ErbB-3 to its C-terminal tail and imply that the flanking kinase domain has lost catalytic activity in order to restrain the relatively potent signaling capability of the C-terminus.

Lipid signaling in CD95-mediated apoptosis

Ceramides play an important role mediating different cell responses such as proliferation, differentiation, growth arrest and apoptosis. They are released upon sphingomyelin hydrolysis which occurs after triggering of a number of cell surface receptors including CD95. Ceramide generation also regulates glycosphingolipid and ganglioside metabolism. In particular, ganglioside GD3 biosynthesis represents an important event for the progression of apoptotic signals generated by CD95 and mediated by ceramide in hematopoietic cells.

Modulation of neuronal and recombinant GABAA receptors by redox reagents.

1. The functional role played by the postulated disulphide bridge in gamma-aminobutyric acid type A (GABAA) receptors and its susceptibility to oxidation and reduction were studied using recombinant (murine receptor subunits expressed in human embryonic kidney cells) and rat neuronal GABAA receptors in conjunction with whole-cell and single channel patch-clamp techniques. 2. The reducing agent dithiothreitol (DTT) reversibly potentiated GABA-activated responses (IGABA) of alpha1beta1 or alpha1beta2 receptors while the oxidizing reagent 5, 5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) caused inhibition. Redox modulation of IGABA was independent of GABA concentration, membrane potential and the receptor agonist and did not affect the GABA EC50 or Hill coefficient. The endogenous antioxidant reduced glutathione (GSH) also potentiated IGABA in alpha1beta2 receptors, while both the oxidized form of DTT and glutathione (GSSG) caused small inhibitory effects. 3. Recombinant receptors composed of alpha1beta1gamma2S or alpha1beta2gamma2S were considerably less sensitive to DTT and DTNB. 4. For neuronal GABAA receptors, IGABA was enhanced by flurazepam and relatively unaffected by redox reagents. However, in cultured sympathetic neurones, nicotinic acetylcholine-activated responses were inhibited by DTT whilst in cerebellar granule neurones, NMDA-activated currents were potentiated by DTT and inhibited by DTNB. 5. Single GABA-activated ion channel currents exhibited a conductance of 16 pS for alpha1beta1 constructs. DTT did not affect the conductance or individual open time constants determined from dwell time histograms, but increased the mean open time by affecting the channel open probability without increasing the number of cell surface receptors. 6. A kinetic model of the effects of DTT and DTNB suggested that the receptor existed in equilibrium between oxidized and reduced forms. DTT increased the rate of entry into reduced receptor forms and also into desensitized states. DTNB reversed these kinetic effects. 7. Our results indicate that GABAA receptors formed by alpha and beta subunits are susceptible to regulation by redox agents. Inclusion of the gamma2 subunit in the receptor, or recording from some neuronal GABAA receptors, resulted in reduced sensitivity to DTT and DTNB. Given the suggested existence of alphabeta subunit complexes in some areas of the central nervous system together with the generation and release of endogenous redox compounds, native GABAA receptors may be subject to regulation by redox mechanisms.

2] Fluorescence techniques for measuring ion channel activity

Publisher Summary Changes in intracellular free calcium concentration ([Ca 2+ ] i ) play a crucial role in cellular physiology. A number of cell surface receptors and channels are known to regulate [Ca 2+ ] i through different molecular mechanisms. Therefore, the functional and pharmacologic properties of many of these cell surface receptors and ion channels can be studied effectively by measuring changes in [Ca 2+ ] i in intact cells. For drug discovery efforts, several ion channel and receptor systems have been targeted that play different roles in neuronal physiology and pathophysiology. These molecular targets include voltage- and ligand-gated ion channels: the human neuronal voltage-gated calcium channels (VGCCs), ligand-gated nicotinic acetylcholine receptor channels (NAChRs), ionotropic N -methyl-D-aspartic acid (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainate-type excitatory amino acid receptor (EAA) channels. All of these channels mediate elevation of [Ca 2+ ] i via Ca 2+ influx from the extracellular medium upon depolarization or activation by agonist. This chapter describes the experimental methods with particular emphasis on the validation of the assay for human VGCCs, NAChRs, and NMDA receptors.

Lipid and glycolipid mediators in CD95-induced apoptotic signaling.

Ceramides play an important role mediating different cell responses such as proliferation, differentiation, growth arrest and apoptosis. They are released upon sphingomyelin hydrolysis which occurs after triggering of a number of cell-surface receptors including CD95. Ceramides generation also regulates glycosphingolipids and gangliosides metabolism. In particular, ganglioside GD3 biosynthesis may represent an important event for the progression of apoptotic signals generated by CD95 in hematopoietic cells.

Chemokine receptor CCR2 expression and monocyte chemoattractant protein-1-mediated chemotaxis in human monocytes. A regulatory role for plasma LDL.

The subendothelial accumulation of macrophage-derived foam cells is one of the hallmarks of atherosclerosis. The recruitment of monocytes to the intima requires the interaction of locally produced chemokines with specific cell surface receptors, including the receptor (CCR2) for monocyte chemoattractant protein-1 (MCP-1). We have previously reported that monocyte CCR2 gene expression and function are effectively downregulated by proinflammatory cytokines. In this study we identified low density lipoprotein (LDL) as a positive regulator of CCR2 expression. Monocyte CCR2 expression was dramatically increased in hypercholesterolemic patients compared with normocholesterolemic controls. Similarly, incubation of human THP-1 monocytes with LDL induced a rapid increase in CCR2 mRNA and protein. By 24 hours the number of cell surface receptors was doubled, causing a 3-fold increase in the chemotactic response to MCP-1. The increase in CCR2 expression and chemotaxis was promoted by native LDL but not by oxidized LDL. Oxidized LDL rapidly downregulated CCR2 expression, whereas reductively methylated LDL, which does not bind to the LDL receptor, had only modest effects on CCR2 expression. A neutralizing anti-LDL receptor antibody prevented the effect of LDL, suggesting that binding and internalization of LDL were essential for CCR2 upregulation. The induction of CCR2 expression appeared to be mediated by LDL-derived cholesterol, because cells treated with free cholesterol also showed increased CCR2 expression. These data suggest that elevated plasma LDL levels in conditions such as hypercholesterolemia enhance monocyte CCR2 expression and chemotactic response and potentially contribute to increased monocyte recruitment to the vessel wall in chronic inflammation and atherogenesis.

Byssinosis: role of polymer length on the effect of tannin on the airway beta-adrenergic receptor.

Tannin, isolated from cotton bracts and implicated in the pathogenesis of byssinosis, inhibits isoproterenol and forskolin-stimulated cAMP release from airway cells in part by decreasing cell surface beta-adrenergic receptor number and uncoupling the beta-adrenergic receptor from its stimulatory G-protein (Gs) and in part by inhibiting adenylyl cyclase activity. We have hypothesized that cotton tannin, because of its long polymer length, interacts with the hydrophobic binding pocket of the beta-adrenergic receptor and alters beta-adrenergic receptor binding and Gs coupling. In these studies, tannins of three different polymer lengths and molecular masses were isolated from cotton bracts using sequential Amicon ultrafiltration [molecular mass > 10, 000 (YM10 retentate), 1,000-10,000 (YM10 filtrate), and 1,000-5,000 Da (YM2 retentate)]. The YM10 retentate (25 microg/ml) decreased chloride secretion (Jnet = 1.11 +/- 0.28 (control) to 0.59 +/- 0.18 microEq/cm2.h, p < 0.05, n = 6), decreased cell surface beta-adrenergic receptor number (18.0 +/- 1.8 (control) to 10.6 +/- 0.9 fmol/mg protein, p < 0.02, n = 4), and inhibited forskolin-stimulated cAMP release (5,254 +/- 1,290 (control) to 2, 968 +/- 620 pmol/mg protein, p < 0.01, n = 8). In contrast, neither the YM10 filtrate nor the YM2 retentate had any effect on net chloride secretion, beta-adrenergic cell surface receptor number, or forskolin-stimulated cAMP release. We conclude that polymer length is essential for the effect of tannin on the beta-adrenergic receptor and on adenylyl cyclase.

A single amino acid substitution in transmembrane helix VI results in overexpression of the human GnRH receptor.

Construction of constitutively active mutants of the GnRH receptor, a member of the G-protein coupled receptor superfamily, would facilitate investigation of the mechanism of receptor activation. Point mutations were introduced in the human GnRH receptor in positions corresponding to those which caused constitutive activity in other G-protein coupled receptors. The effects of these mutations on ligand binding, receptor intracellular signaling and receptor expression were determined. Wild type and mutated receptor cDNAs were expressed in COS-1 cells. Basal and agonist-stimulated inositol phosphate production and ligand binding were determined. In addition, receptor mRNA levels, cell surface receptor stability and rate of internalization were measured. Although none of the mutant receptors exhibited constitutive activity, mutation of Phe-2 72 in transmembrane helix VI to Leu increased cell surface receptor numbers, with unchanged affinities for radiolabeled agonist, superagonist and antagonist peptides compared with wild type receptor. The cell surface receptor stability and rate of internalization were similar for wild type and F272L GnRH receptors. Thus a single amino acid mutation in transmembrane helix VI causes an increase in cell surface receptor numbers, which appears to result from an increased rate of receptor protein translation, processing or insertion into membranes.

Identification of ceramide targets in interleukin-1 and tumor necrosis factor-α signaling in mesangial cells

An increasing number of cell-surface receptors have been shown to trigger sphingomyelin turnover and generation of the lipid signaling molecule ceramide. Ceramide plays a role in mediating cellular responses as diverse as inflammation, differentiation, gene expression, growth suppression, and apoptosis. A radioiodinated, photoaffinity-labeling analog of ceramide ([125I]TID-ceramide) was used to identify downstream signaling targets of ceramide. Ceramide was found to bind specifically to and activate protein kinase c-Raf, leading to subsequent activation of the extracellular signal-regulated kinase (ERK) module in mesangial cells. We found also that ceramide binds to and differentially modulates the activity of distinct protein kinase C isoenzymes. These data are discussed in the context of interleukin 1beta-induced inflammatory gene expression in mesangial cells.

Infection of glial cells by the human polyomavirus JC is mediated by an N-linked glycoprotein containing terminal alpha(2-6)-linked sialic acids.

The human JC polyomavirus (JCV) is the etiologic agent of the fatal central nervous system (CNS) demyelinating disease progressive multifocal leukoencephalopathy (PML). PML typically occurs in immunosuppressed patients and is the direct result of JCV infection of oligodendrocytes. The initial event in infection of cells by JCV is attachment of the virus to receptors present on the surface of a susceptible cell. Our laboratory has been studying this critical event in the life cycle of JCV, and we have found that JCV binds to a limited number of cell surface receptors on human glial cells that are not shared by the related polyomavirus simian virus 40 (C. K. Liu, A. P. Hope, and W. J. Atwood, J. Neurovirol. 4:49-58, 1998). To further characterize specific JCV receptors on human glial cells, we tested specific neuraminidases, proteases, and phospholipases for the ability to inhibit JCV binding to and infection of glial cells. Several of the enzymes tested were capable of inhibiting virus binding to cells, but only neuraminidase was capable of inhibiting infection. The ability of neuraminidase to inhibit infection correlated with its ability to remove both alpha(2-3)- and alpha(2-6)-linked sialic acids from glial cells. A recombinant neuraminidase that specifically removes the alpha(2-3) linkage of sialic acid had no effect on virus binding or infection. A competition assay between virus and sialic acid-specific lectins that recognize either the alpha(2-3) or the alpha(2-6) linkage revealed that JCV preferentially interacts with alpha(2-6)-linked sialic acids on glial cells. Treatment of glial cells with tunicamycin, but not with benzyl N-acetyl-alpha-D-galactosaminide, inhibited infection by JCV, indicating that the sialylated JCV receptor is an N-linked glycoprotein. As sialic acid containing glycoproteins play a fundamental role in mediating many virus-cell and cell-cell recognition processes, it will be of interest to determine what role these receptors play in the pathogenesis of PML.

Molecules in focus: The c-Cbl oncoprotein

Cbl has emerged as a novel signal transducing protein downstream of a number of cell surface receptors coupled to tyrosine kinases. Identified as the protein product of the c- cbl proto-oncogene, the cellular homolog to the transforming gene of a murine retrovirus, Cbl comprises an N-terminal transforming region (Cbl-N), which contains a phosphotyrosine binding (PTB) domain, and a C-terminal modular region (Cbl-C) containing a RING finger motif, a large proline-rich region and a leucine zipper. Deletion of Cbl-C or small deletions N-terminal to the RING finger render Cbl oncogenic, whereas wild type Cbl is non-transforming, even if overexpressed. Cbl serves as a substrate of both receptor and non-receptor tyrosine kinases, and binds to adaptor proteins Grb2, Crk and the p85 subunit of PI-3-kinase. Additionally, both Caenorhabditis elegans and Drosophila Cbl homologs, SLI-1 and D-Cbl, respectively, have been identified as negative regulators of the LET-23/DER receptor tyrosine kinases. Finally, oncogenic mutants of Cbl, when expressed in fibroblasts, upregulate the signaling cascade downstream of the platelet-derived growth factor receptor α in a Cbl-PTB domain-dependent manner. Together, these findings position Cbl as a central player in the regulation of tyrosine kinase signaling pathways. Identification of the Cbl-PTB domain binding motifs on tyrosine kinases and elucidation of the mechanisms of Cbl's negative regulatory effect may provide a new avenue to control tyrosine kinases for therapeutic purposes.

Involvement of receptor cycling and receptor reserve in resensitization of muscarinic responses in SH-SY5Y human neuroblastoma cells.

Preexposure of SH-SY5Y cells to the muscarinic agonist carbachol caused a rapid desensitization of subsequent carbachol-stimulated intracellular Ca2+ responses and a slower decrease in the number of receptors at the plasma membrane. Desensitization (to 30% of the control response) was maximal after 1 min of exposure to agonist, whereas the number of cell surface receptors reached a minimum (33% of control) only after 5 min. Following agonist washout, the recovery of response was complete within 12 min, whereas the recovery of surface receptor number reached a plateau at 65% of control after 30 min. Treatment with inhibitors of endocytosis (concanavalin A) or recycling (nigericin) did not affect rapid desensitization but did decrease resensitization, suggesting that receptor cycling is involved in resensitization. Experiments with the irreversible antagonist propylbenzilylcholine mustard demonstrated that the receptor reserve for the Ca2+ response to 1 mM carbachol is approximately 50%. Removal of this receptor reserve led to a decrease in the rate of resensitization. We propose that the existence of a receptor reserve might explain the poor correlation between functional response and surface receptor number, and that one of its roles might be to permit rapid resensitization after a significant agonist-induced decrease in surface receptor number. The purpose of receptor cycling might be to allow dephosphorylation (and reactivation) of receptors that have become phosphorylated (and inactivated) in response to agonist stimulation, because the protein phosphatase inhibitor calyculin A significantly reduced resensitization.

Interleukin 10 increases CCR5 expression and HIV infection in human monocytes.

The immunosuppressive and antiinflammatory cytokine interleukin (IL) 10 selectively upregulates the expression of the CC chemokine receptors CCR5, 2, and 1 in human monocytes by prolonging their mRNA half-life. IL-10-stimulated monocytes display an increased number of cell surface receptors for, and better chemotactic responsiveness to, relevant agonists than do control cells. In addition, IL-10-stimulated monocytes are more efficiently infected by HIV BaL. This effect was associated to the enhancement of viral entry through CCR5. These data add support to an emerging paradigm in which pro- and antiinflammatory molecules exert reciprocal and opposing influence on chemokine agonist production and receptor expression.

Afr1p regulates the Saccharomyces cerevisiae alpha-factor receptor by a mechanism that is distinct from receptor phosphorylation and endocytosis.

The alpha-factor pheromone receptor activates a G protein signaling pathway that induces the conjugation of the yeast Saccharomyces cerevisiae. Our previous studies identified AFR1 as a gene that regulates this signaling pathway because overexpression of AFR1 promoted resistance to alpha-factor. AFR1 also showed an interesting genetic relationship with the alpha-factor receptor gene, STE2, suggesting that the receptor is regulated by Afr1p. To investigate the mechanism of this regulation, we tested AFR1 for a role in the two processes that are known to regulate receptor signaling: phosphorylation and down-regulation of ligand-bound receptors by endocytosis. AFR1 overexpression diminished signaling in a strain that lacks the C-terminal phosphorylation sites of the receptor, indicating that AFR1 acts independently of phosphorylation. The effects of AFR1 overexpression were weaker in strains that were defective in receptor endocytosis. However, AFR1 overexpression did not detectably influence receptor endocytosis or the stability of the receptor protein. Instead, gene dosage studies showed that the effects of AFR1 overexpression on signaling were inversely proportional to the number of receptors. These results indicate that AFR1 acts independently of endocytosis, and that the weaker effects of AFR1 in strains that are defective in receptor endocytosis were probably an indirect consequence of their increased receptor number caused by the failure of receptors to undergo ligand-stimulated endocytosis. Analysis of the ligand binding properties of the receptor showed that AFR1 overexpression did not alter the number of cell-surface receptors or the affinity for alpha-factor. Thus, Afr1p prevents alpha-factor receptors from activating G protein signaling by a mechanism that is distinct from other known pathways.