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.
Highlights
The human lutropin receptor3 is a G protein-coupled receptor (GPCR) that plays a central role in reproductive physiology
To determine whether hLHR self-association could be detected in living cells, we examined whether specific BRET2 signals would be observed in cells co-transfected hLHR(wt)Renilla luciferase (Rluc) and hLHR(wt)-GFP2
Little or no detectable BRET2 ratios were detected under those conditions, suggesting that the BRET2 signals observed between hLHR(wt)Rluc and hLHR(wt)-GFP2 are because of the physical interaction between the hLHR portions of the fusion proteins
Summary
The human lutropin receptor (hLHR) is a G protein-coupled receptor (GPCR) that plays a central role in reproductive physiology. Agonist-occupied wildtype hLHR or hLHR CAMs activate the Gs, Gi/o, and Gq/11 families of G proteins [5, 6], with the primary actions of the hLHR being mediated by Gs. A large body of work published in recent years supports the concept that GPCRs can form self-associated dimers and higher ordered oligomers within cells 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] In this latter study it was shown that under basal conditions the hLHR physically self-associates into complexes of sizes consistent with dimers and higher ordered oligomers of the receptor. These results could be explained by an agonistdependent induction of hLHR dimerization, it may reflect a stabilization of preformed receptor dimers/oligomers to detergent solubilization when they are occupied by hCG
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