Two Defective Heterozygous Luteinizing Hormone Receptors Can Rescue Hormone Action

Lee, ChangWoo; Ji, Inhae; Ryu, Kyeong‐Sik; Song, Yong Sang; Conn, P. Michael; Ji, Tae H.

doi:10.1074/jbc.m111818200

Cited by 63 publications

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References 40 publications

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“…Evidence pointing toward this goes back to as early as the 1980s when data from equilibrium sedimentation of detergent-solubilized LHR and radiation inactivation of LHRs on gonadal cells suggested that the LHR may be present in a dimeric or oligomeric form (31,32). More recently, functional complementation studies further suggested the dimerization of the LHR (33,34). Direct evidence in support of dimerization of the LHR has been obtained using fluorescent resonance energy transfer (FRET) (35)(36)(37)(38) as well as our studies demonstrating specific co-immunoprecipitation of differentially tagged forms of the LHR (39).…”

mentioning

confidence: 66%

“…To examine the potential homodimerization/oligomerization of a signaling inactive hLHR mutant, we initially tested hLHR(K605R), which had previously been reported (using an alternate nomenclature as K583R) to be signaling inactive (33,34). However, when compared with cells expressing the same cell surface density of WT hLHR, we found K605R responded to hCG with ϳ30% of the maximal cAMP response of the WT hLHR.…”

Section: Resultsmentioning

confidence: 99%

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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

Guan

et al. 2009

Journal of Biological Chemistry

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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.

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confidence: 66%

Section: Resultsmentioning

confidence: 99%

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

Guan

et al. 2009

Journal of Biological Chemistry

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“…We selected two LHR mutant receptors for their inability to bind the ligand (LH or hCG) or to transduce signaling after ligand binding. The first mutant receptor (LHR LH− ) harbored an inactivating Cys 22 to Ala 22 mutation in the ligand binding extracellular domain (35,36). The second mutant (LHR cAMP− ) contained a deletion of TM helices 6 and 7 in exon 11 (amino acids deleted from Val 553 to Ala 689 ), and it was chosen because of involvement of the deleted region in G protein coupling and second messenger generation (37).…”

Section: Resultsmentioning

confidence: 99%

“…There is recent functional evidence that the class A GPCRs for glycoprotein hormones [i.e., of LH/choriongonadotropin (hCG), FSH, and TSH] could transduce their signal in cultured cells as di/ oligomers by trans-activation (8,10,14,35) (Fig. 1B), henceforth termed intermolecular cooperation.…”

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confidence: 99%

Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

Rivero-Müller

Chou

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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189

141

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G protein–coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling.

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“…However, for the luteinizing hormone receptor, which also has a long N-terminal domain that binds the ligand, partial reconstitution of function has been achieved by co-expression of distinct pairs of mutants (15)(16). Furthermore, in recent studies co-expression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescued hormone-activated cAMP production (17).…”

Section: Mutation Of a Highly Conserved Leucine In Intracellular Loopmentioning

confidence: 99%

Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

Carrillo

Pediani

Milligan

2003

Journal of Biological Chemistry

106

118

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The concept that G protein-coupled receptors (GPCRs) 1 exist as dimers or higher order oligomers has moved rapidly from hypothesis to being widely accepted (1-4). A range of approaches has contributed to this understanding. This includes the ability to co-immunoprecipitate differentially epitopetagged forms of a GPCR from cells in which they are co-expressed, and, in intact cells, the application of a number of resonance energy transfer-based techniques. However, the role of dimerization in function and the mechanisms then responsible for initiation of signal transduction by the dimer have been more recalcitrant to analysis. Significant progress in this area has recently been achieved for the class C GPCRs. These contain both a long extracellular N-terminal domain, to which agonist ligands bind, and the prototypic seven transmembrane (TM) helix bundle architecture that is the common feature of all GPCR families (for review, see Ref. 5). The functional ␥-aminobutyric acid, type b receptor is a hetero-dimer of two distinct gene products in which trafficking to the plasma membrane requires interaction between the partner polypeptides (6 -9). This indicates that a key role of dimerization is achieving appropriate cellular localization. This is also true for the class A rhodopsin-like GPCRs because non-functional, truncated splice variants can restrict plasma membrane delivery of fulllength GPCRs and, thus, limit their function (10 -12). Chimeric class C GPCRs consisting of the extracellular domain of one GPCR and the TM and intracellular elements of a second, closely related GPCR or in which the intracellular loops of dimer partners are exchanged have provided strong evidence that the mechanism of action of the dimer involves transactivation (13)(14); that is, ligand binding to one element of the dimer results in activation of G protein produced by the other GPCR within the dimer. Again, the ␥-aminobutyric acid, type b receptor has been particularly informative in this regard as only one of the two gene products that forms the dimer is able to bind the agonist ␥-aminobutyric acid. Equivalent systems are not available for the majority of class A GPCRs. However, for the luteinizing hormone receptor, which also has a long N-terminal domain that binds the ligand, partial reconstitution of function has been achieved by co-expression of distinct pairs of mutants (15-16). Furthermore, in recent studies co-expression of a mutant receptor defective in hormone binding and another mutant defective in signal generation rescued hormone-activated cAMP production (17).

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Two Defective Heterozygous Luteinizing Hormone Receptors Can Rescue Hormone Action

Cited by 63 publications

References 40 publications

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

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

Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

Dimers of Class A G Protein-coupled Receptors Function via Agonist-mediated Trans-activation of Associated G Proteins

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