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

Abstract

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.

Fig. 1.

Demonstration of intermolecular cooperation and di/oligomerization on binding- and signaling-deficient LHR mutants in cultured cells. (A and B) Schematic presentation of intramolecular (cis) and intermolecular (trans) activation of GPCRs. (A) When a hormone (green) binds to its WT receptor, the occupied receptor activates itself to generate a signal(s) (5). (B) Alternatively, a GPCR complexed with hormone may activate another GPCR molecule, as evidenced by intermolecular activation of signaling-deficient mutant (LHR^cAMP− with red connecting loops) by binding-deficient mutant (LHR^LH− with red extracellular domain) when both mutants are coexpressed in a cell (33, 35, 36). (C–E) Cell culture experiments on ligand binding and cAMP generation of the LHR mutants. (C) Binding-deficient receptor (LHR^LH−; red line) was incapable of displaying specific binding of [¹²⁵I]-hCG in the presence of increasing concentrations of unlabeled hCG (0–10 nM). In contrast, WT (black line) and signaling-deficient LHR (LHR^cAMP−; blue line) bound [¹²⁵I]-hCG specifically and with similar apparent affinity. (D) WT LHR (black line) produced cAMP in response to hCG stimulation. However, neither the signaling-deficient (LHR^cAMP−, blue line) nor the binding-deficient (LHR^LH−, superimposed with the former) mutant produced cAMP. (E) When both (LHR^LH− and LHR^cAMP−) mutants were coexpressed in HEK-293 cells, cAMP production was partially restored in response to hCG (purple line) as compared to WT LHR (black line). One of three experiments with similar results transfecting with BAC-LHR clones is shown. Each point is the mean ± SD of triplicate incubations. Experiments with cDNA clones produced similar results.

Fig. 2.

LHR cellular localization and di/oligomerization. (A) Cell surface expression. HEK-293 cells expressing tagged-LHRs (HA-WT, HA-LHR^LH−, or FLAG-LHR^cAMP−) were immunostained using anti-HA or anti-FLAG antibodies, respectively, and secondary antibodies labeled with fluorescent dyes, and analyzed by confocal microscopy. All three receptors (WT and mutants) could be detected on the cell surface as shown on nonpermeabilized cells (Upper), while some receptor immunoreactivity was detected in the ER of permeabilized cells (Lower). This proves that the WT and mutant receptors are transported to the cell surface. (B and C) Dimerization/oligomerization of mutant mLHRs. HEK-293 cells were transiently transfected with FLAG-tagged signaling-deficient (FLAG-LHR^cAMP−) and HA-tagged binding-deficient (HA-LHR^LH−) mutant constructs. Lysates were prepared and subjected to immunoprecipitation (IP) with anti-FLAG antibody. Thereafter, the immunoprecipitates were resolved by SDS/PAGE under reducing conditions, and immunoblots (IB) were probed with either anti-FLAG antibody (B) or anti-HA antibody (C). Both monomeric and higher molecular weight LHR complexes were detected. These data indicate that FLAG-LHR^cAMP− mutant interacts with HA-LHR^LH− mutant. The lanes are: 1, control vector (pcDNA); 2, HA-LHR^LH−; 3, FLAG-LHR^−cAMP; 4, combined lysates from separately FLAG-LHR^−cAMP and HA-LHR^−LH transfected cells; 5, coexpressed FLAG-LHR^−cAMP + HA-LHR^−LH; 6, coexpressed FLAG-LHR^cAMP− + HA-β2-AR (different experiment with identical conditions).

Fig. 3.

Expression of the LHR^cAMP− and LHR^LH− BAC transgenes in male mice. (A) Expression of mRNA of the transgenes (LHR^cAMP− and LHR^LH−) in different tissues of TG mice as analyzed by RT-PCR, showing strong specific expression in gonads (product sizes 559 and 960 bp, respectively) and weak expression in brain. (B) Specific [¹²⁵I]-hCG binding to testis homogenates of WT, LuRKO, LHR^cAMP−, and LHR^LH− mice (n = three per group), as well as, in a separate binding assay, WT (n = 4) and LHR^{LH−/cAMP−} (n = 2) mice (C). Each bar is the mean + SD. Different letters above the bars in B indicate that these levels differ significantly (P at least <0.05).

Fig. 4.

Gonadal and genital phenotypes: macroscopic and microscopic appearance of the WT and mutant male mice. (Top) Testes and accessory sex organs (from left to right) of LuRKO mice (A) and mice expressing in the LuRKO background LHR^LH− (B), LHR^cAMP− (C), both transgenes (LHR^{LH−/ cAMP−}) (D), and WT mice (E). (Middle) Immunofluorescence of the reporter genes (eCFP, green, corresponding to LHR^LH−; RFP, red, corresponding to LHR^cAMP−) specifically expressed in Leydig cells (LC). (Bottom) Merged pictures of the two reporter genes, showing coexpression of the two transgenes in LC of LHR^{LH−/cAMP−} mice. (Scale bars: Top, 1 cm; Middle and Bottom, 25 μm).

Fig. 5.

Testicular histology and serum hormone levels of the WT and mutant mice. (A) LuRKO mice. (B–D) Mice expressing in the LuRKO background LHR^LH−, LHR^cAMP−, or both transgenes (LHR^{LH−/cAMP−}), respectively. (E) WT mice. LuRKO mice and the inactivating mutants alone display the same histology with Leydig cell hypoplasia, narrow seminiferous tubules, and spermatogenesis arrested at the round spermatid stage. When both deficient receptors were coexpressed in the LuRKO background, testicular histology was indistinguishable from WT males. (Scale bar: 50 μm.) (F) Serum LH (filled bars) and testosterone (open bars) in, from left to right, WT, LuRKO, LHR^LH−, LHR^cAMP−, and LHR^{LH−/cAMP−} mice. Each bar denotes the mean ± SD of measurements from at least four mice. Different letters above the bars indicate that these levels differ significantly (P at least <0.05).