M C Gershengorn

The National Institute of Diabetes and Digestive and Kidney Diseases, Maryland, United States

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Publications (87)475.65 Total impact

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    L Ikonomou · E Geras-Raaka · B M Raaka · M C Gershengorn ·
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    ABSTRACT: Previously, we characterized human islet-derived precursor cells (hIPCs) as mesenchymal stem cells that migrate out from islets in vitro and can differentiate into functional islet-like structures following proliferative expansion. Here, we investigate the role of beta-catenin signalling in derivation and proliferation of hIPCs. Localization of beta-catenin was performed using confocal microscopy. Expression levels of beta-catenin target genes were measured by quantitative real-time polymerase chain reaction. Loss-of-function studies were performed using specific short interfering RNAs. Immunostaining of islet outgrowths revealed translocation of beta-catenin from plasma membranes in intact islets to the nucleus in cells migrating out. There were no nuclear beta-catenin-positive cells in intact islets whereas between 35% and 70% of cells in established hIPC cultures exhibited nuclear beta-catenin. Transcripts for beta-catenin target genes were increased in hIPCs compared to those in islets. Beta-catenin translocated to the cell membrane when hIPCs formed epithelial cell clusters. In proliferating hIPCs, there was a strong correlation between markers of proliferation and nuclear beta-catenin. Treatment of hIPCs with the glycogen synthase kinase-3beta inhibitor (2'Z,3'E)-6-Bromoindirubin-3'-oxime increased intracellular beta-catenin but reduced nuclear beta-catenin, and was associated with reduced cell proliferation. Finally, knockdown of beta-catenin decreased beta-catenin target gene expression and hIPC proliferation. These results support a functional role for beta-catenin during proliferation of hIPCs and suggest that activated beta-catenin signalling may also be important during hIPC derivation from islets.
    Cell Proliferation 07/2008; 41(3):474-91. DOI:10.1111/j.1365-2184.2008.00527.x · 3.12 Impact Factor
  • A O Colson · M C Gershengorn ·
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    ABSTRACT: 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.
    Mini Reviews in Medicinal Chemistry 03/2006; 6(2):221-6. DOI:10.2174/138955706775476019 · 2.90 Impact Factor
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    Y Sun · X Lu · M C Gershengorn ·
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    ABSTRACT: 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.
    Journal of Molecular Endocrinology 05/2003; 30(2):87-97. · 3.08 Impact Factor
  • J P Couty · E Geras-Raaka · B B Weksler · M C Gershengorn ·
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    ABSTRACT: 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.
    Journal of Biological Chemistry 10/2001; 276(36):33805-11. DOI:10.1074/jbc.M104631200 · 4.57 Impact Factor
  • S Harder · X Lu · W Wang · F Buck · M C Gershengorn · T O Bruhn ·
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    ABSTRACT: 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.
    Endocrinology 04/2001; 142(3):1188-94. · 4.50 Impact Factor
  • M Lupu-Meiri · R B Silver · AH Simons · M C Gershengorn · Y Oron ·
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    ABSTRACT: 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.
    Journal of Biological Chemistry 04/2001; 276(10):7122-8. DOI:10.1074/jbc.M006359200 · 4.57 Impact Factor
  • H H Ho · N Ganeshalingam · A Rosenhouse-Dantsker · R Osman · M C Gershengorn ·
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    ABSTRACT: 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.
    Journal of Biological Chemistry 02/2001; 276(2):1376-82. DOI:10.1074/jbc.M007885200 · 4.57 Impact Factor
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    F Buck · W Wang · S Harder · C Brathwaite · T O Bruhn · M C Gershengorn ·
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    ABSTRACT: 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.
    Endocrinology 11/2000; 141(10):3717-22. · 4.50 Impact Factor
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    B F O'Dowd · D K Lee · W Huang · T Nguyen · R Cheng · Y Liu · B Wang · M C Gershengorn · S R George ·
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    ABSTRACT: 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.
    Molecular Endocrinology 02/2000; 14(1):183-93. · 4.02 Impact Factor
  • H H Ho · D Du · M C Gershengorn ·
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    ABSTRACT: 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.
    Journal of Biological Chemistry 11/1999; 274(44):31327-32. DOI:10.1074/jbc.274.44.31327 · 4.57 Impact Factor
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    W Wang · M C Gershengorn ·
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    ABSTRACT: 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.
    Endocrinology 11/1999; 140(10):4916-9. DOI:10.1210/en.140.10.4916 · 4.50 Impact Factor
  • A O Colson · J H Perlman · A Jinsi-Parimoo · D R Nussenzveig · R Osman · M C Gershengorn ·
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    ABSTRACT: We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.
    Molecular Pharmacology 01/1999; 54(6):968-78. · 4.13 Impact Factor
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    M C Gershengorn · E Geras-Raaka · A Varma · I Clark-Lewis ·
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    ABSTRACT: 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.
    Journal of Clinical Investigation 11/1998; 102(8):1469-72. DOI:10.1172/JCI4461 · 13.22 Impact Factor
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    YM Sun · R P Millar · H Ho · M C Gershengorn · N Illing ·
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    ABSTRACT: We report on the cloning of the full-length complementary DNA for the chicken TRH receptor. Although the TRH receptor has been cloned from several mammalian species, this is the first report from another vertebrate class. The ligand binding pocket, which is situated in the transmembrane helices of the mouse and rat TRH receptors, is completely conserved in the chicken receptor. Pharmacological studies (receptor binding and signaling) employing several TRH analogs revealed that there are no significant differences between the chicken and mouse receptors. These findings show that there have been considerable evolutionary constraints on TRH receptor structure and function. Several truncated forms of the chicken TRH receptor that appear to retain a part of an intron and are truncated in the putative third intracellular loop were also cloned, but were nonfunctional. This study provides a useful tool for further studies on the roles of TRH in avian growth and TSH regulation.
    Endocrinology 09/1998; 139(8):3390-8. DOI:10.1210/en.139.8.3390 · 4.50 Impact Factor
  • J H Perlman · AO Colson · W Wang · K Bence · R Osman · M C Gershengorn ·
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    ABSTRACT: The roles of conserved residues in transmembrane helices (TMs) of G protein-coupled receptors have not been well established. A computer-generated model of the thyrotropin-releasing hormone receptor (TRH-R) indicated that conserved Asp-71 (TM-2) could interact with conserved asparagines 316 (TM-7) and 43 (TM-1). To test this model, we constructed mutant TRH-Rs containing polar or alanine substitutions of these residues. The maximal activities of N43A and N316A TRH-Rs were diminished, whereas D71A (Perlman, J. H., Nussenzveig, D. R., Osman, R., and Gershengorn, M. C. (1992) J. Biol. Chem. 267, 24413-24417) and N43A/N316A TRH-Rs were inactive. Computer models of D71A and N43A/N316A TRH-Rs show similar changes from native TRH-R in their TM bundle conformations. The inactivity and the similarity of the computer models of D71A and N43A/N316A TRH-Rs are consistent with the idea that Asp-71 bridges Asn-43 and Asn-316 and suggest that activity is critically dependent on these interactions. The conservation of these residues suggests these specific interactions involving TMs 1, 2, and 7 may be structurally important for all members of the rhodopsin/beta-adrenergic receptor subfamily of G protein-coupled receptors.
    Journal of Biological Chemistry 06/1997; 272(18):11937-42. DOI:10.1074/jbc.272.18.11937 · 4.57 Impact Factor
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    A Jinsi-Parimoo · M C Gershengorn ·
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    ABSTRACT: The native TRH receptor (TRH-R), which is a G protein-coupled receptor that signals via the phosphoinositide transduction pathway, has been assumed to be inactive in the absence of agonist. In contrast, a mutant mouse TRH-R (C335Stop TRH-R) was shown previously to exhibit constitutive (or agonist-independent) signaling activity. In this report, we measured signaling activity of TRH-Rs using a protein kinase C-responsive reporter gene instead of formation of inositol phosphate second messenger molecules. Using this more sensitive system, we show that native mouse TRH-Rs exhibit agonist-independent signaling activity that is directly proportional to the number of receptors expressed in COS-1 cells and is inhibited by negative antagonist benzodiazepine drugs. As expected, the basal signaling activity of native TRH-Rs is lower than C335Stop TRH-Rs. Constitutive activity of native TRH-Rs is not peculiar to COS-1 cells in which receptor density is markedly elevated, because it can also be demonstrated in Madin Darby canine kidney cells stably expressing mouse TRH-Rs and GH4C1 cells endogenously expressing rat TRH-Rs. These findings support the thesis that native TRH-Rs oscillate between active and inactive states. We suggest that demonstration of constitutive activity of native receptors may depend on the sensitivity of the signaling assay employed.
    Endocrinology 05/1997; 138(4):1471-5. DOI:10.1210/en.138.4.1471 · 4.50 Impact Factor
  • S J Dermer · D P Cohen · C N Thaw · D R Nussenzveig · M C Gershengorn ·
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    ABSTRACT: Overexpression of native and epitope-tagged human calcitonin (CT) receptors (hCTR-2) in COS-1 cells was performed to permit identification of the receptor protein and begin studies of receptor turnover. Data obtained with immunological techniques and cross-linking of radiolabeled salmon CT ([125I]sCT) revealed two forms of hCTR-2 in transfected cells: a larger, mature cell surface receptor (apparent size, 81 kDa) and a smaller, intracellular form (apparent size, 66 kDa). These conclusions are based on the following observations. 1) Only the larger hCTR-2 was visualized by cell surface [125I]sCT binding, whereas both species were identified by [125I]sCT binding to cell lysates. 2) Immunofluorescence studies with antibodies directed against the epitope confirmed the presence of cell surface and intracellular hCTR-2s; there were apparently many more receptors intracellularly than on the cell surface. 3) Both hCTR-2 forms were changed to a similar size of approximately 57-60 kDa by deglycosylation with endoglycosidase F; this size is consistent with that predicted by the amino acid sequence. Metabolic studies with radioactive amino acids labeled only the intracellular form. This immature form exhibited a rapid half-life of 30 min. We conclude that overexpression of native and epitope-tagged hCTR-2s in COS-1 cells leads to their intracellular retention and rapid degradation.
    Endocrinology 01/1997; 137(12):5502-8. · 4.50 Impact Factor
  • D P Cohen · D R Nussenzveig · M C Gershengorn ·
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    ABSTRACT: The reported binding affinities of cloned human calcitonin receptors (hCTRs) for salmon calcitonin (sCT) vary over a wide range. Because the greatest differences were between values estimated in association binding versus competition binding experiments, we considered the possibility that this variation was due to differences in the affinities of hCTRs for moniodinated sCT (I-sCT) and sCT. We found that I-sCT competed with 125I-sCT for binding with 9.3 +/- 0.58 times the apparent affinity of sCT in COS-1 and Madin Darby canine kidney (MDCK) cells expressing cloned hCTRs. I-sCT was 25 times more potent than sCT in stimulating cAMP formation in MDCK cells expressing hCTRs. These data demonstrate that monoiodinated sCT interacts more avidly with hCTRs than sCT with a consequent enhanced potency in stimulating second messenger formation. Therefore, as I-sCT (or 125I-sCT) binds with different affinity to hCTRs than sCT, affinity measurements must be performed with I-sCT (or 125I-sCT) alone.
    Endocrinology 11/1996; 137(10):4507-10. · 4.50 Impact Factor
  • L Laakkonen · W Li · J H Perlman · F Guarnieri · R Osman · K D Moeller · M C Gershengorn ·
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    ABSTRACT: Thyrotropin-releasing hormone (TRH) is a tripeptide (< Glu-His-Pro-NH2) that signals through a G protein-coupled receptor. TRH is a highly flexible molecule that can assume many conformations in solution. To attempt to delineate the biologically active conformation of TRH, we synthesized a pair of conformationally restricted cyclohexyl/Ala2-TRH analogues. The diastereomeric analogues use a lactam ring to restrict two of the six free torsional angles of TRH and constrain the X-Pro-NH2 peptide bond to trans. Unrestricted cyclohexyl/Ala2-TRH exhibited a 650-fold lower affinity than TRH for TRH receptor and was 430-fold less potent than TRH in stimulating inositol phosphate second messenger formation. One diastereomer exhibited higher affinity and potency than the unrestricted analogue despite the presence of the methylene bridge and fused ring, whereas the other showed lower affinity and potency. Computer simulations predicted that the positions of the cyclohexyl/Ala2 and Pro-NH2 moieties relative to < glutamate were different in the two analogues and that the conformation of the higher affinity analogue is different from that of trans-TRH in solution but is superimposable on that of trans-TRH found in a model of the TRH/TRH receptor complex. These experimental findings identify a favored relative position of < glutamate and Pro-NH2 in the more active conformation of two diastereomeric analogues of TRH and provide independent support for the model of the TRH/TRH receptor complex.
    Molecular Pharmacology 06/1996; 49(6):1092-6. · 4.13 Impact Factor
  • C Yeaman · M Heinflink · E Falck-Pedersen · E Rodriguez-Boulan · M C Gershengorn ·
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    ABSTRACT: Information concerning the molecular sorting of G protein-coupled receptors in polarized epithelial cells is limited. Therefore, we have expressed the receptor for thyrotropin-releasing hormone (TRH) in Madin-Darby canine kidney (MDCK) cells by adenovirus-mediated gene transfer to determine its distribution in a model cell system and to begin analyzing the molecular information responsible for its distribution. Equilibrium binding of [methyl-3H]TRH to apical and basolateral surfaces of polarized MDCK cells reveals that TRH receptors are expressed predominantly (>80%) on the basolateral cell surface. Receptors undergo rapid endocytosis following agonist binding; up to 80% are internalized in 15 min. A mutant receptor missing the last 59 residues, C335Stop, is poorly internalized (<10%) but is nevertheless basolaterally expressed (>85%). A second mutant TRH receptor, delta218-263, lacks essentially all of the third intracellular loop and is not coupled to G proteins on binding agonist. This receptor internalizes TRH approximately half as efficiently as wild-type TRH receptors but is nevertheless strongly polarized to the basolateral surface (>90%). These results indicate that molecular sequences responsible for basolateral accumulation of TRH receptors can be segregated from signals for ligand-induced receptor endocytosis and coupling to heterotrimeric G proteins.
    The American journal of physiology 04/1996; 270(3 Pt 1):C753-62. · 3.28 Impact Factor

Publication Stats

3k Citations
475.65 Total Impact Points


  • 2006-2008
    • The National Institute of Diabetes and Digestive and Kidney Diseases
      Maryland, United States
  • 2003
    • National Institutes of Health
      • National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
      Bethesda, MD, United States
  • 1988-2001
    • Cornell University
      • Department of Medicine
      Итак, New York, United States
  • 1985-2001
    • Weill Cornell Medical College
      • Department of Medicine
      New York, New York, United States
  • 1990-1994
    • Tel Aviv University
      • Department of Physiology and Pharmacology
      Tel Aviv, Tel Aviv, Israel
  • 1978-1984
    • CUNY Graduate Center
      New York, New York, United States