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M C Gershengorn

Publications and source records attributed to M C Gershengorn.

At least 19 recordsLinked to original sources
PubMed2006

Thyrotropin-releasing hormone analogs.

Thyrotropin releasing hormone (TRH: pyroglutamic acid-histidine-prolineamide) regulates the activity of cells in the anterior pituitary and within the central and peripheral nervous systems. TRH, which has been the subject of much research over the past three decades, exerts its effects by acting through class A G-protein coupled receptors. The recent discovery of a second receptor subtype has generated an interest in the discovery of receptor subtype-selective TRH analogs. In this review, we describe advances in the development of TRH analogs and in the understanding of their mechanism of interaction with TRH receptors. We also describe the recent breakthrough in the identification of analogs that bind selectively at TRH-R2.

Animals↗
PubMed2003

Thyrotropin-releasing hormone receptors -- similarities and differences.

Thyrotropin-releasing hormone (TRH) initiates its effects by interacting with cell-surface membrane receptors. Two G protein-coupled receptors for TRH, TRH receptor type 1 (TRH-R1) and TRH receptor type 2 (TRH-R2), have been cloned from mammals. In this review, we compare TRH-R1 and TRH-R2 with regard to their tIssue distribution, binding affinities for TRH and TRH analogs, basal and activated signaling activities and characteristics of internalization. TRH-R1 and TRH-R2 are distributed differently in the brain and peripheral tIssues, but exhibit indistinguishable binding affinities for TRH and TRH analogs. Although they both can be stimulated by TRH to similar maximal signaling levels, TRH-R2 exhibits higher basal signaling activity and is more rapidly internalized than TRH-R1. These differences in signaling and internalization properties are probably important in the distinct parts that TRH-R1 and TRH-R2 may play in mammalian physiology.

Animals↗
PubMed2001

Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor signals through multiple pathways in endothelial cells.

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) encodes a chemokine-like G protein-coupled receptor (KSHV-GPCR) that is implicated in the pathogenesis of Kaposi's sarcoma (KS). Since endothelial cells appear to be targets for the virus, we developed an in vitro mouse lung endothelial cell model in which KSHV-GPCR is stably expressed and KSHV-GPCR signaling was studied. In mouse lung endothelial cells: 1) KSHV-GPCR does not exhibit basal signaling through the phosphoinositide-specific phospholipase C pathway but inositol phosphate production is stimulated by growth-related oncogene alpha (Gro-alpha) via a pertussis toxin (PTX)-insensitive pathway; 2) KSHV-GPCR signals basally through a PTX-sensitive pathway leading to a lowering of intracellular cAMP level that can be lowered further by Gro alpha and increased by interferon gamma-inducible protein 10; 3) KSHV-GPCR stimulates phosphatidylinositol 3-kinase via a PTX-insensitive mechanism; and 4) KSHV-GPCR activates nuclear factor-kappa B (NF-kappa B) by a PTX-sensitive G beta gamma subunit-mediated pathway. These data show that KSHV-GPCR couples to at least two G proteins and initiates signaling via at least three cascades in endothelial cells thereby increasing the complexity of regulation of endothelial cell function by KSHV-GPCR that may occur during viral infection.

Adenylyl Cyclases↗
PubMed2001

Charged residues at the intracellular boundary of transmembrane helices 2 and 3 independently affect constitutive activity of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor.

Because charged residues at the intracellular ends of transmembrane helix (TMH) 2 and TMH3 of G protein-coupled receptors (GPCRs) affect signaling, we performed mutational analysis of these residues in the constitutively signaling Kaposi's sarcoma-associated herpesvirus GPCR (KSHV-GPCR). KSHV-GPCR contains the amino acid sequence Val-Arg-Tyr rather than the Asp/Glu-Arg-Tyr ((D/E)RY) motif at the intracellular end of TMH3. Mutation of Arg-143 to Ala (R143A) or Gln (R143Q) abolished constitutive signaling whereas R143K exhibited 50% of the basal activity of KSHV-GPCR. R143A was not stimulated by agonist, whereas R143Q was stimulated by growth-related oncogene-alpha, and R143K, similar to KSHV-GPCR, was stimulated further. These findings show that Arg-143 is critical for signal generation in KSHV-GPCR. In other GPCRs, Arg in this position may act as a signaling switch by movement of its sidechain from a hydrophilic pocket in the TMH bundle to a position outside the bundle. In rhodopsin, the Arg of Glu-Arg-Tyr interacts with the adjacent Asp to constrain Arg outside the TMH bundle. V142D was 70% more active than KSHV-GPCR, suggesting that an Arg residue, which is constrained outside the bundle by interacting with Asp-142, leads to a receptor that signals more actively. Because the usually conserved Asp in the middle of TMH2 is not present in KSHV-GPCR, we tested whether Asp-83 at the intracellular end of TMH2 was involved in signaling. D83N and D83A were 110 and 190% more active than KSHV-GPCR, respectively. The double mutant D83A/V142D was 510% more active than KSHV-GPCR. That is, cosubstitutions of Asp-83 by Ala and Val-142 by Asp act synergistically to increase basal signaling. A model of KSHV-GPCR predicts that Arg-143 interacts with residues in the TMH bundle and that the sidechain of Asp-83 does not interact with Arg-143. These data are consistent with the hypothesis that Arg-143 and Asp-83 independently affect the signaling activity of KSHV-GPCR.

Amino Acid Sequence↗
PubMed2001

Minireview: Insights into G protein-coupled receptor function using molecular models.

G protein-coupled receptors (GPCRs) represent the largest family of signal-transducing molecules known. They convey signals for light and many extracellular regulatory molecules. GPCRs have been found to be dysfunctional/dysregulated in a growing number of human diseases and have been estimated to be the targets of more than 30% of the drugs used in clinical medicine today. Thus, understanding how GPCRs function at the molecular level is an important goal of biological research. In order to understand function at this level, it is necessary to delineate the 3D structure of these receptors. Recently, the 3D structure of rhodopsin has been resolved, but in the absence of experimentally determined 3D structures of other GPCRs, a powerful approach is to construct a theoretical model for the receptor and refine it based on experimental results. Computer-generated models for many GPCRs have been constructed. In this article, we will review these studies. We will place the greatest emphasis on an iterative, bi-directional approach in which models are used to generate hypotheses that are tested by experimentation and the experimental findings are, in turn, used to refine the model. The success of this approach is due to the synergistic interaction between theory and experiment.

Amino Acid Sequence↗
PubMed2001

Regulator of G protein signaling 4 suppresses basal and thyrotropin releasing-hormone (TRH)-stimulated signaling by two mouse TRH receptors, TRH-R(1) and TRH-R(2).

We cloned the mouse TRH receptor type 2 (mTRH-R2) gene, which is 92% identical with rat TRH-R2 and 50% identical with mTRH-R1 at the amino acid level, and identified an intron within the coding sequence that is not present in the TRH-R1 gene structure. Similar to its rat homolog, mTRH-R2 binds TRH with an affinity indistinguishable from mTRH-R1, signals via the phosphoinositide pathway like mTRH-R1, but exhibits a higher basal signaling activity than mTRH-R1. We found that regulator of G protein signaling 4 (RGS4), which differentially inhibits signaling by other receptors that couple to Gq, inhibits TRH-stimulated signaling via mTRH-R1 and mTRH-R2 to similar extents. In contrast, other RGS proteins including RGS7, RGS9, and GAIP had no effect on signaling by mTRH-R1 or mTRH-R2 demonstrating the specificity of RGS4 action. Interestingly, RGS4 markedly inhibited basal signaling by mTRH-R2. Inhibition of basal signaling of mTRH-R2 by RGS4 suggests that modulation of agonist-independent signaling may be an important mechanism of regulation of G protein-coupled receptor activity under normal physiologic circumstances.

Amino Acid Sequence↗
PubMed2000

Constitutive signaling by Kaposi's sarcoma-associated herpesvirus G-protein-coupled receptor desensitizes calcium mobilization by other receptors.

We coexpressed Kaposi's sarcoma-associated herpesvirus G protein-coupled receptors (KSHV-GPCRs) with thyrotropin-releasing hormone (TRH) receptors or m1-muscarinic-cholinergic receptors in Xenopus oocytes and in mammalian cells. In oocytes, KSHV-GPCR expression resulted in pronounced (81%) inhibition (heterologous desensitization) of Ca(2+)-activated chloride current responses to TRH and acetylcholine. Similar inhibitions of cytoplasmic free Ca(2+) responses to TRH were observed in human embryonic kidney HEK 293 EM cells and in mouse pituitary AtT20 cells. Further study of oocytes showed that this inhibition was partially reversed by interferon-gamma-inducible protein 10 (IP-10), an inverse agonist of KSHV-GPCR. The basal rate of (45)Ca(2+) efflux in oocytes expressing KSHV-GPCRs was 4.4 times greater than in control oocytes, and IP-10 rapidly inhibited increased (45)Ca(2+) efflux. In the absence of IP-10, growth-related oncogene alpha caused a further 2-fold increase in (45)Ca(2+) efflux. In KSHV-GPCR-expressing oocytes, responses to microinjected inositol 1,4,5-trisphosphate were inhibited by 74%, and this effect was partially reversed by interferon-gamma-inducible protein 10. Treatment with thapsigargin suggested that the pool of calcium available for mobilization by TRH was decreased in oocytes coexpressing KSHV-GPCRs. These results suggest that constitutive signaling by KSHV-GPCR causes heterologous desensitization of responses mediated by other receptors, which signal via the phosphoinositide/calcium pathway, which is caused by depletion of intracellular calcium pools.

Acetylcholine↗
PubMed2000

Dynorphin stimulates corticotropin release from mouse anterior pituitary AtT-20 cells through nonopioid mechanisms.

Dynorphin (Dyn) peptides were previously shown to increase plasma corticotropin (ACTH) in the ovine fetus, but the site of its action remains unclear. In the present study, Dyn A(1-17) was found to stimulate ACTH release from mouse anterior pituitary tumor AtT-20 cells in a dose-dependent manner. Naloxone did not block the effect of Dyn A(1-17) and the selective kappa-opioid receptor agonist U50488H did not stimulate ACTH release. Dyn A(2-17), a degradative peptide fragment that does not bind to opioid receptors, also stimulated ACTH release from AtT-20 cells. Although the nonopioid effects of Dyn have previously been attributed to N-methyl-D-aspartate (NMDA) receptors, the ACTH-releasing effects of Dyn A(1-17) in AtT-20 cells were not affected by co-administration of NMDA receptor antagonist LY235959. The ACTH response to Dyn A(1-17) could not be blocked by alpha-helical CRH (CRH antagonist) and was additive with a maximal stimulatory dose of CRH, suggesting different mechanisms of action. These results show that the release of ACTH by Dyn A(1-17) in AtT-20 cells is not mediated by kappa-opioid receptors or by the NMDA receptor.

Adrenocorticotropic Hormone↗
PubMed2000

Juxtamembrane regions in the third intracellular loop of the thyrotropin-releasing hormone receptor type 1 are important for coupling to Gq.

Juxtamembrane residues in the putative third intracellular (I3) loops of a number of G protein-coupled receptors (GPCRs) have been shown to be important for coupling to G proteins. According to standard hydropathy analysis, the I3 loop of the mouse TRH receptor type 1 (mTRH-R1) is composed of 51 amino acids from position-213 to position-263. We constructed deletion and site-specific I3 loop TRH-R mutants and studied their binding and TRH-stimulated signaling activities. As expected, the effects of these mutations on TRH binding were small (less than 5-fold decreases in affinity). No effect on TRH-stimulated signaling activity was found in a mutant receptor in which the I3 loop was shortened to 16 amino acids by deleting residues from Asp-226 to Ser-260. In contrast, mutants with deletions from Asp-222 to Ser-260 or from Asp-226 to Gln-263 exhibited reduced TRH-stimulated signaling. In the region near transmembrane helix 6, single site-specific substitution of either Arg-261 or Lys-262 by neutral glutamine had little effect on signaling, but mutant TRH-Rs that were substituted by glutamine at both basic residues exhibited reduced TRH-stimulated activity. The reduced signaling activity of this doubly substituted mutant was reversed by over expressing the a subunit of Gq. These data demonstrate that the juxtamembrane regions in the TRH-R I3 loop are important for coupling to Gq.

Amino Acid Sequence↗
PubMed2000

TRH-R2 exhibits similar binding and acute signaling but distinct regulation and anatomic distribution compared with TRH-R1.

TRH (thyroliberin) is a tripeptide (pGlu-His-ProNH2) that signals via G protein-coupled receptors. Until recently, only a single receptor for TRH was known (TRH-R1), but two groups identified a second receptor, TRH-R2. We independently discovered TRH-R2. Using an extensive set of TRH analogs, we found no differences in TRH-R1 and TRH-R2 binding or in acute stimulation of signaling. TRH-R2 was more rapidly internalized upon binding TRH and exhibited a greater level of TRH-induced down-regulation than TRH-R1. During prolonged exposure to TRH, cells expressing TRH-R2 exhibited a lower level of gene induction than cells expressing TRH-R1. TRH-R2 receptor mRNA was present in very discrete nuclei and regions of rat brain. A major mRNA transcript for TRH-R2 was seen in the cerebral cortex, pons, thalamus, hypothalamus, and midbrain with faint bands found in the striatum and pituitary. The extensive distribution of TRH-R2 in the brain suggests that it mediates many of the known functions of TRH that are not transduced by TRH-R1. The variations in agonist-induced internalization and down-regulation/desensitization, and anatomic distribution of TRH-R2 compared with TRH-R1, suggest important functional differences between the two receptors.

Amino Acid Sequence↗
PubMed1999

The N terminus of Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor is necessary for high affinity chemokine binding but not for constitutive activity.

Kaposi's sarcoma-associated herpesvirus (KSHV) contains a gene encoding a G protein-coupled receptor (KSHV-GPCR) that is homologous to mammalian chemokine receptors. KSHV-GPCR signals constitutively (in an agonist-independent manner) via the phosphoinositide-inositol 1,4,5-trisphosphate pathway. Because it has been proposed that the N terminus (N-TERM) of other GPCRs may act as tethered agonists, we determined whether the N-TERM of KSHV-GPCR is necessary for constitutive signaling activity or ligand binding, or both. We show that replacement of the entire N-TERM of KSHV-GPCR with those of two other GPCRs, deletion of residues within the N-TERM, and disruption of a putative disulfide bond that may hold the N-TERM in close proximity to extracellular loop 3 do not affect constitutive signaling activity but decrease chemokine binding. There were differences in the effects of mutation of the N-TERM on binding of the chemokines growth-related oncogene alpha, which is an agonist, and interferon-gamma-inducible protein-10, which is an inverse agonist. The effects on chemokine binding were accompanied by changes in chemokine regulation of KSHV-GPCR signaling. We conclude that the N-TERM is not necessary for constitutive KSHV-GPCR signaling, i.e. the N-TERM is not a tethered agonist, but plays a crucial role in binding of chemokine ligands and of chemokine regulation of KSHV-GPCR signaling.

Animals↗
PubMed1999

Glycosylation is important for binding to human calcitonin receptors.

Human calcitonin receptor (hCTR) subtypes contain three or four potential Asn-linked glycosylation sites in their extracellular amino termini. The role of glycosylation in hCTR function has not been identified, but it has been suggested that inhibition of glycosylation does not affect binding or signaling. To determine the role of glycosylation in hCTR biology, we studied the effects of inhibition of glycosylation and of substitution of Asn residues that are potential glycosylation sites. Native and mutated hCTRs were studied after transient expression in monkey kidney COS-1 cells. Tunicamycin, administered as part of a treatment protocol that inhibited glycosylation of all expressed receptors, decreased salmon calcitonin (sCT) binding affinities and signaling potencies at hCTRs with three or four potential glycosylation sites. In hCTR3, which contains three potential glycosylation sites at positions 26, 78, and 83, site-specific substitution of Asn-26 by Ala had no effect on sCT binding affinity or potency, whereas substitution of Asn-78 or Asn-83 lowered sCT affinity and potency. A mutant hCTR3 in which all three Asn residues were substituted with Ala exhibited no high-affinity sCT binding and potencies of several calcitonin analogues that were more than 100-fold lower than that of native hCTR3. Our data show that glycosylation is important for high-affinity binding and potency of calcitonin analogues at hCTRs.

Alanine↗
PubMed1999

Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin-releasing hormone receptor: role of cellular calcium and protein kinase C.

1. C335Stop is a constitutively active mutant of the TRH receptor (TRH-R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH-R, we studied cellular Ca2+ concentrations ([Ca2+]i) in AtT20 cells stably transfected with C335Stop TRH-R cDNA, or Ca2+-activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH-R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization. 2. Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca2+]i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca2+]i and a decrease in TRH response amplitude. 3. CDE abolished heterologous desensitization induced by C335Stop TRH-R on muscarinic m1-receptor (ml-R) co-expressed in Xenopus oocytes. 4. Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+]i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH-Rs. 5. Chelerythrine, a specific inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the effect of chelerythrine. 6. Down-regulation of PKC by chronic exposure to phorbol 12-myristate 13-acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH. 7. Western analysis indicated that the alpha subtype of protein kinase C was down-regulated in cells expressing C335Stop TRH-Rs. Following a 5 min exposure to PMA, the residual alphaPKC translocated to the particular fraction. 8. We propose that cells expressing the constitutively active mutant TRH-R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca2+]i and PKC.

Animals↗
PubMed1999

Rat TRH receptor type 2 exhibits higher basal signaling activity than TRH receptor type 1.

Two types of rat TRH receptor (TRH-R1 and TRH-R2) have been identified and shown previously to exhibit similar binding and stimulated signaling activity via the phosphoinositide-calcium transduction pathway. Since mouse TRH-R1 exhibits basal (or constitutive or ligand-independent) signaling activity, we compared basal signaling by TRH-R1 and TRH-R2. Basal signaling was measured as receptor-mediated reporter gene induction via different transcription factors. We found that TRH-R2 exhibited higher basal signaling activity than TRH-R1 via pathways mediated by transcription factors AP-1, Elk-1 and CREB. Furthermore, CREB-mediated transcription was directly dependent on the level of TRH-R2 expression and was inhibited by midazolam, a specific inverse agonist of basal TRH-R signaling. Since TRH-R1 and TRH-R2 exhibit distinct anatomic distributions in the rat, it is possible that TRH ligand-independent signaling is more important in tissues/cells in which TRH-R2 is expressed and less important in tissues in which TRH-R1 is found.

Animals↗
PubMed1998

Kaposi's sarcoma-associated herpesvirus (KSHV) chemokine vMIP-II and human SDF-1alpha inhibit signaling by KSHV G protein-coupled receptor.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes two proteins that are similar to human CC chemokines and a G protein-coupled receptor (KSHV-GPCR) that is constitutively active. KSHV-GPCR binds a number of human CXC and CC chemokines. We showed that interferon gamma-inducible protein-10 (IP-10), a human CXC chemokine, inhibits KSHV-GPCR signaling (Geras-Raaka et al., J. Exp. Med. 188, 405-408, 1998). Here we show that viral monocyte inflammatory protein-II (vMIP-II), one of the KSHV-encoded CC chemokines, and stromal cell-derived factor 1alpha (SDF-1alpha), a human CXC chemokine that blocks infection by human immunodeficiency virus-type 1, inhibit KSHV-GPCR signaling also. If KSHV-GPCR signaling is involved in viral pathogenesis, then these chemokines may affect the course of Kaposi's sarcoma and primary effusion lymphoma.

Chemokine CXCL12↗
PubMed1998

Thyrotropin releasing hormone analogs: a building block approach to the construction of tetracyclic peptidomimetics.

A building block based approach was used to synthesize a pair of tetracyclic peptidomimetics that constrain all but one of the rotational degrees of freedom of the hypothalamic tripeptide hormone thyroliberin. One of the analogs bound to the thyroliberin endocrine receptor (TRH-R) with an affinity greater than that of an analog without constraints. The tetracyclic peptidomimetics were found to be partial agonists for the TRH-R receptor.

Animals↗
PubMed1998

Chemokines activate Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor in mammalian cells in culture.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, a virus that appears to be involved in the pathogenesis of Kaposi's sarcoma and primary effusion lymphomas, encodes a G protein-coupled receptor (KSHV-GPCR) that exhibits constitutive signaling. In this report, we show that two chemokines, interleukin 8 (IL-8) and growth-related protein-alpha, activate KSHV-GPCR over constitutive levels. Moreover, as with human receptors, the integrity of the ELR motif of these chemokines is required for activation of KSHV-GPCR. Other residues that are required for IL-8 binding to human chemokine receptors CXCR1 and CXCR2 are important for KSHV-GPCR activation also. Thus, it appears that the ELR binding site and other key domains of ELR chemokine activation have been preserved in the virus KSHV-GPCR. The results suggest that KSHV-GPCR originated from CXCR1 or CXCR2 and that activation of KSHV-GPCR by endogenous chemokines may affect the pathobiology of KSHV infection in humans.

3T3 Cells↗