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PMID: 19147490 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

Bioluminescence resonance energy transfer studies reveal constitutive dimerization of the human lutropin receptor and a lack of correlation between receptor activation and the propensity for dimerization.

The Journal of biological chemistry ·Vol. 284 ·No. 12 ·2009-03-20 ·Pages 7483-94

Guan R, Feng X, Wu X, Zhang M, Zhang X, Hébert TE, Segaloff DL

Abstract

Previous studies from our laboratory using co-immunoprecipitation techniques suggested that the human lutropin receptor (hLHR) constitutively self-associates into dimers/oligomers and that agonist treatment of cells either increased hLHR dimerization/oligomerization and/or stabilized hLHR dimers/oligomers to detergent solubilization (Tao, Y. X., Johnson, N. B., and Segaloff, D. L. (2004) J. Biol. Chem. 279, 5904-5914). In this study, bioluminescence resonance energy transfer (BRET(2)) analyses confirmed that the hLHR constitutively self-associates in living cells. After subcellular fractionation, hLHR dimers/oligomers were detected in both the plasma membrane and endoplasmic reticulum (ER). Further evidence supporting the constitutive formation of hLHR dimer/oligomers in the ER is provided by data showing homodimerization of misfolded hLHR mutants that are retained in the ER. These mutants, when co-expressed with wild-type receptor, are shown by BRET(2) to heterodimerize, accounting for their dominant-negative effects on cell surface receptor expression. Hormone desorption assays using intact cells demonstrate allosterism between hLHR protomers, indicating functional cell surface hLHR dimers. However, quantitative BRET(2) analyses in intact cells indicate a lack of effect of agonist on the propensity of the hLHR to dimerize. Using purified plasma membranes, human chorionic gonadotropin was similarly observed to have no effect on the BRET(2) signal. An examination of the propensity for constitutively active and signaling inactive hLHR mutants to dimerize further showed no correlation between dimerization and the activation state of the hLHR. Taken altogether, our data suggest that hLHR dimers/oligomers are formed early in the biosynthetic pathway in the ER, are constitutively expressed on the plasma membrane, and are not affected by the activation state of the hLHR.

MeSH Terms
Cell Line Cell Membrane/genetics,metabolism Chorionic Gonadotropin/genetics,metabolism Dimerization Endoplasmic Reticulum/genetics,metabolism Gene Expression Regulation/physiology Humans Luminescent Measurements Protein Folding Receptors, LH/agonists,genetics,metabolism
Chemicals
Chorionic Gonadotropin Receptors, LH
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Guan Rongbin
Department of Molecular Biophysics and Physiology, The Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242, USA.
Feng Xiuyan
Wu Xueqing
Zhang Meilin
Zhang Xuesen
Hébert Terence E
Segaloff Deborah L
References (79)
79 references, click to expand
  1. Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity.
    EMBO J. 2005 Jun 1;24(11):1954-64 PMID: 15889138
  2. Negative cooperativity of glutamate binding in the dimeric metabotropic glutamate receptor subtype 1.
    J Biol Chem. 2004 Aug 20;279(34):35526-34 PMID: 15199056
  3. Chemokine receptor homo- or heterodimerization activates distinct signaling pathways.
    EMBO J. 2001 May 15;20(10):2497-507 PMID: 11350939
  4. A molecular dissection of the glycoprotein hormone receptors.
    Trends Biochem Sci. 2004 Mar;29(3):119-26 PMID: 15003269
  5. Two defective heterozygous luteinizing hormone receptors can rescue hormone action.
    J Biol Chem. 2002 May 3;277(18):15795-800 PMID: 11859079
  6. A constitutively active somatic mutation of the human lutropin receptor found in Leydig cell tumors activates the same families of G proteins as germ line mutations associated with Leydig cell hyperplasia.
    Endocrinology. 2003 Sep;144(9):3872-8 PMID: 12933660
  7. Insight into mutation-induced activation of the luteinizing hormone receptor: molecular simulations predict the functional behavior of engineered mutants at M398.
    Mol Endocrinol. 2004 Jun;18(6):1499-508 PMID: 15016840
  8. Dimerization of G-protein-coupled receptors: roles in signal transduction.
    Cell Signal. 2004 Feb;16(2):175-86 PMID: 14636888
  9. Cooperativity manifest in the binding properties of purified cardiac muscarinic receptors.
    J Biol Chem. 1995 Sep 22;270(38):22488-99 PMID: 7673239
  10. Heterodimerization of mu and delta opioid receptors: A role in opiate synergy.
    J Neurosci. 2000 Nov 15;20(22):RC110 PMID: 11069979
  11. Detecting and imaging protein-protein interactions during G protein-mediated signal transduction in vivo and in situ by using fluorescence-based techniques.
    Cell Biochem Biophys. 2006;45(1):85-109 PMID: 16679566
  12. Brief report: testicular and ovarian resistance to luteinizing hormone caused by inactivating mutations of the luteinizing hormone-receptor gene.
    N Engl J Med. 1996 Feb 22;334(8):507-12 PMID: 8559204
  13. Evidence for negative binding cooperativity within CCR5-CCR2b heterodimers.
    Mol Pharmacol. 2005 Feb;67(2):460-9 PMID: 15509716
  14. Luteinizing hormone receptors are self-associated in slowly diffusing complexes during receptor desensitization.
    Mol Endocrinol. 2001 Apr;15(4):534-42 PMID: 11266505
  15. Luteinizing hormone receptors are self-associated in the plasma membrane.
    Endocrinology. 2000 Dec;141(12):4518-23 PMID: 11108263
  16. Quantitative assessment of beta 1- and beta 2-adrenergic receptor homo- and heterodimerization by bioluminescence resonance energy transfer.
    J Biol Chem. 2002 Nov 22;277(47):44925-31 PMID: 12244098
  17. Theoretical study on mutation-induced activation of the luteinizing hormone receptor.
    J Mol Biol. 2000 Mar 10;296(5):1333-51 PMID: 10698637
  18. Transfected cells express mostly the intracellular precursor of the lutropin/choriogonadotropin receptor but this precursor binds choriogonadotropin with high affinity.
    Biochemistry. 1998 Jan 13;37(2):664-72 PMID: 9425089
  19. Involvement of the amino terminus of the B(2) receptor in agonist-induced receptor dimerization.
    J Biol Chem. 1999 Sep 10;274(37):26079-84 PMID: 10473556
  20. Emerging role of homo- and heterodimerization in G-protein-coupled receptor biosynthesis and maturation.
    Trends Pharmacol Sci. 2005 Mar;26(3):131-7 PMID: 15749158
  21. AT1-receptor heterodimers show enhanced G-protein activation and altered receptor sequestration.
    Nature. 2000 Sep 7;407(6800):94-8 PMID: 10993080
  22. Allosteric properties of G protein-coupled receptor oligomers.
    Pharmacol Ther. 2007 Sep;115(3):410-8 PMID: 17655934
  23. HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization.
    Proc Natl Acad Sci U S A. 2000 Mar 28;97(7):3388-93 PMID: 10725362
  24. Assessment of mechanistic proposals for the binding of agonists to cardiac muscarinic receptors.
    Biochemistry. 1986 Nov 4;25(22):6995-7008 PMID: 3801407
  25. Potential Leydig cell mitogenic signals generated by the wild-type and constitutively active mutants of the lutropin/choriogonadotropin receptor (LHR).
    Mol Cell Endocrinol. 2007 Jan 2;260-262:244-8 PMID: 17055151
  26. Constitutive activation of the LH receptor is associated with an alteration in the conformation of the ectodomain.
    Mol Cell Endocrinol. 2002 Aug 30;194(1-2):211-5 PMID: 12242044
  27. Orexin-1 receptor-cannabinoid CB1 receptor heterodimerization results in both ligand-dependent and -independent coordinated alterations of receptor localization and function.
    J Biol Chem. 2006 Dec 15;281(50):38812-24 PMID: 17015451
  28. Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET).
    Nat Methods. 2006 Mar;3(3):165-74 PMID: 16489332
  29. Gonadotropin binding and stimulation of steroidogenesis in Leydig tumor cells.
    Proc Natl Acad Sci U S A. 1978 Jan;75(1):99-102 PMID: 203945
  30. Allosteric modulation of heterodimeric G-protein-coupled receptors.
    Trends Pharmacol Sci. 2007 Dec;28(12):615-20 PMID: 18022255
  31. Dimerization of the extracellular calcium-sensing receptor (CaR) on the cell surface of CaR-transfected HEK293 cells.
    J Biol Chem. 1998 Sep 4;273(36):23605-10 PMID: 9722601
  32. Oxytocin and vasopressin V1a and V2 receptors form constitutive homo- and heterodimers during biosynthesis.
    Mol Endocrinol. 2003 Apr;17(4):677-91 PMID: 12554793
  33. Functional and structural characterization of rhodopsin oligomers.
    J Biol Chem. 2006 Apr 28;281(17):11917-22 PMID: 16495215
  34. Ligand-dependent inhibition of oligomerization at the human thyrotropin receptor.
    J Biol Chem. 2002 Nov 22;277(47):45059-67 PMID: 12223484
  35. Constitutive agonist-independent CCR5 oligomerization and antibody-mediated clustering occurring at physiological levels of receptors.
    J Biol Chem. 2002 Sep 20;277(38):34666-73 PMID: 12089144
  36. The chemokine SDF-1alpha triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway.
    FASEB J. 1999 Oct;13(13):1699-710 PMID: 10506573
  37. Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations.
    J Biol Chem. 2007 Aug 31;282(35):25527-39 PMID: 17609213
  38. The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor.
    J Biol Chem. 2005 Jul 15;280(28):26169-76 PMID: 15908694
  39. Cardiac muscarinic receptors. Cooperativity as the basis for multiple states of affinity.
    Biochemistry. 1997 Jun 17;36(24):7361-79 PMID: 9200684
  40. Serotonin 5-HT2C receptor homodimer biogenesis in the endoplasmic reticulum: real-time visualization with confocal fluorescence resonance energy transfer.
    J Biol Chem. 2006 Sep 15;281(37):27109-16 PMID: 16857671
  41. Receptors for dopamine and somatostatin: formation of hetero-oligomers with enhanced functional activity.
    Science. 2000 Apr 7;288(5463):154-7 PMID: 10753124
  42. Hypersensitization of the Orexin 1 receptor by the CB1 receptor: evidence for cross-talk blocked by the specific CB1 antagonist, SR141716.
    J Biol Chem. 2003 Jun 27;278(26):23731-7 PMID: 12690115
  43. The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2.
    Proc Natl Acad Sci U S A. 1999 Mar 30;96(7):3628-33 PMID: 10097088
  44. A model for constitutive lutropin receptor activation based on molecular simulation and engineered mutations in transmembrane helices 6 and 7.
    J Biol Chem. 2002 Aug 30;277(35):32202-13 PMID: 12070159
  45. Lutropin receptor function: insights from natural, engineered, and computer-simulated mutations.
    IUBMB Life. 2001 Mar;51(3):149-55 PMID: 11547916
  46. Calcium-sensing receptor dimerizes in the endoplasmic reticulum: biochemical and biophysical characterization of CASR mutants retained intracellularly.
    Hum Mol Genet. 2006 Jul 15;15(14):2200-9 PMID: 16740594
  47. Cis- and trans-activation of hormone receptors: the LH receptor.
    Mol Endocrinol. 2002 Jun;16(6):1299-308 PMID: 12040016
  48. Simultaneous activation of the delta opioid receptor (deltaOR)/sensory neuron-specific receptor-4 (SNSR-4) hetero-oligomer by the mixed bivalent agonist bovine adrenal medulla peptide 22 activates SNSR-4 but inhibits deltaOR signaling.
    Mol Pharmacol. 2006 Aug;70(2):686-96 PMID: 16682504
  49. Methods to monitor the quaternary structure of G protein-coupled receptors.
    FEBS J. 2005 Jun;272(12):2914-25 PMID: 15955052
  50. Roles of G-protein-coupled receptor dimerization.
    EMBO Rep. 2004 Jan;5(1):30-4 PMID: 14710183
  51. Follice-stimulating hormone receptor forms oligomers and shows evidence of carboxyl-terminal proteolytic processing.
    Endocrinology. 2007 May;148(5):1987-95 PMID: 17272391
  52. The lutropin/choriogonadotropin receptor, a 2002 perspective.
    Endocr Rev. 2002 Apr;23(2):141-74 PMID: 11943741
  53. A day in the life of a G protein-coupled receptor: the contribution to function of G protein-coupled receptor dimerization.
    Br J Pharmacol. 2008 Mar;153 Suppl 1:S216-29 PMID: 17965750
  54. BRET analysis of GPCR oligomerization: newer does not mean better.
    Nat Methods. 2007 Jan;4(1):3-4; author reply 4 PMID: 17195017
  55. G protein-coupled receptor dimerisation: molecular basis and relevance to function.
    Biochim Biophys Acta. 2007 Apr;1768(4):825-35 PMID: 17069751
  56. The CXCR1 and CXCR2 receptors form constitutive homo- and heterodimers selectively and with equal apparent affinities.
    J Biol Chem. 2005 Aug 5;280(31):28663-74 PMID: 15946947
  57. Male pseudohermaphroditism due to a homozygous missense mutation of the luteinizing hormone receptor gene.
    Nat Genet. 1995 Feb;9(2):160-4 PMID: 7719343
  58. Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer.
    J Biol Chem. 2001 Apr 20;276(16):12736-43 PMID: 11278883
  59. Incorporation of radiolabeled amino acids into protein subunits of the rat leydig cell gonadotropin receptor: application to the study of receptor structure and turnover.
    Ann N Y Acad Sci. 1984;438:224-36 PMID: 6100014
  60. Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors.
    Mol Endocrinol. 2001 May;15(5):695-703 PMID: 11328852
  61. A role for heterodimerization of mu and delta opiate receptors in enhancing morphine analgesia.
    Proc Natl Acad Sci U S A. 2004 Apr 6;101(14):5135-9 PMID: 15044695
  62. Metabotropic glutamate receptor 5 is a disulfide-linked dimer.
    J Biol Chem. 1996 Nov 8;271(45):28612-6 PMID: 8910492
  63. Naturally occurring mutations of the luteinizing-hormone receptor: lessons learned about reproductive physiology and G protein-coupled receptors.
    Am J Hum Genet. 1999 Oct;65(4):949-58 PMID: 10486313
  64. Constitutive and agonist-dependent self-association of the cell surface human lutropin receptor.
    J Biol Chem. 2004 Feb 13;279(7):5904-14 PMID: 14594799
  65. Characterization of ovarian gonadotropin receptor. Monomer and associated form of the receptor.
    J Biol Chem. 1988 Feb 25;263(6):3046-9 PMID: 3343239
  66. Heterotrimeric G proteins form stable complexes with adenylyl cyclase and Kir3.1 channels in living cells.
    J Cell Sci. 2006 Jul 1;119(Pt 13):2807-18 PMID: 16787947
  67. Membrane organization of luteinizing hormone receptors differs between actively signaling and desensitized receptors.
    J Biol Chem. 2003 Oct 31;278(44):42744-9 PMID: 12930832
  68. Identification of two distinct structural motifs that, when added to the C-terminal tail of the rat LH receptor, redirect the internalized hormone-receptor complex from a degradation to a recycling pathway.
    Mol Endocrinol. 2001 Sep;15(9):1624-35 PMID: 11518811
  69. Intracellularly located misfolded glycoprotein hormone receptors associate with different chaperone proteins than their cognate wild-type receptors.
    Mol Endocrinol. 2004 Jul;18(7):1768-77 PMID: 15105436
  70. Mutations of gonadotropins and gonadotropin receptors: elucidating the physiology and pathophysiology of pituitary-gonadal function.
    Endocr Rev. 2000 Oct;21(5):551-83 PMID: 11041448
  71. An inactivating mutation of the luteinizing hormone receptor causes amenorrhea in a 46,XX female.
    J Clin Endocrinol Metab. 1996 Nov;81(11):3850-4 PMID: 8923827
  72. Homodimerization of the beta2-adrenergic receptor as a prerequisite for cell surface targeting.
    J Biol Chem. 2004 Aug 6;279(32):33390-7 PMID: 15155738
  73. Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer.
    J Cell Biol. 1998 Jul 13;142(1):69-84 PMID: 9660864
  74. G protein-coupled receptor dimers: functional consequences, disease states and drug targets.
    Pharmacol Ther. 2008 Jun;118(3):359-71 PMID: 18486226
  75. Cell-surface targeting of alpha2-adrenergic receptors -- inhibition by a transport deficient mutant through dimerization.
    Cell Signal. 2006 Mar;18(3):318-27 PMID: 15961277
  76. G protein-coupled receptor allosterism and complexing.
    Pharmacol Rev. 2002 Jun;54(2):323-74 PMID: 12037145
  77. An intracellular loop (IL2) residue confers different basal constitutive activities to the human lutropin receptor and human thyrotropin receptor through structural communication between IL2 and helix 6, via helix 3.
    Endocrinology. 2008 Apr;149(4):1705-17 PMID: 18162522
  78. Self-association and raft localization of functional luteinizing hormone receptors.
    Biol Reprod. 2003 Dec;69(6):1765-70 PMID: 12890728
  79. Constitutively-active human LH receptors are self-associated and located in rafts.
    Mol Cell Endocrinol. 2007 Jan 2;260-262:65-72 PMID: 17045393
Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M809150200
Published
2009-03-20
Epub
2009-00-15
Pages
7483-94
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2658044
Subset
IM
Grants
NIDDK NIH HHS · DK068614 · United States
NICHD NIH HHS · HD22196 · United States
External Links
PubMed source DOI: 10.1074/jbc.M809150200 PMC Full Text View journal details
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