Luteinizing hormone receptor ectodomain splice variant misroutes the full-length receptor into a subcompartment of the endoplasmic reticulum

Abstract

The luteinizing hormone receptor (LHR) is a G protein-coupled receptor that is expressed in multiple RNA messenger forms. The common rat ectodomain splice variant is expressed concomitantly with the full-length LHR in tissues and is a truncated transcript corresponding to the partial ectodomain with a unique C-terminal end. Here we demonstrate that the variant alters the behavior of the full-length receptor by misrouting it away from the normal secretory pathway in human embryonic kidney 293 cells. The variant was expressed as two soluble forms of M(r) 52,000 and M(r) 54,000, but although the protein contains a cleavable signal sequence, no secretion to the medium was observed. Only a very small fraction of the protein was able to gain hormone-binding ability, suggesting that it is retained in the endoplasmic reticulum (ER) by its quality control due to misfolding. This was supported by the finding that the variant was found to interact with calnexin and calreticulin and accumulated together with these ER chaperones in a specialized juxtanuclear subcompartment of the ER. Only proteasomal blockade with lactacystin led to accumulation of the variant in the cytosol. Importantly, coexpression of the variant with the full-length LHR resulted in reduction in the number of receptors that were capable of hormone binding and were expressed at the cell surface and in targeting of immature receptors to the juxtanuclear ER subcompartment. Thus, the variant mediated misrouting of the newly synthesized full-length LHRs may provide a way to regulate the number of cell surface receptors.

Figure 1.

Schematic presentation of the rat LHR gene structure and topography of the full-length receptor and its ectodomain splice variant. (A) Exons 1–10 and the 5′ end of exon 11 code for the extracellular domain, whereas the rest of exon 11 encodes the seven transmembrane sequences and the C-terminal intracellular domain of the receptor. The arrow indicates the alternative splice site within exon 11. Cys-knot, cysteine knot; TM, transmembrane. (B) The truncated variant contains the N-terminal cysteine-knot and part of the C-terminal one (dark gray) with a unique C-terminal tail (light gray). The six potential N-glycosylation sites in the ectodomain and cysteine residues (black circles) are indicated.

Figure 2.

The LHRvariant is a soluble protein containing high-mannose-type N-linked oligosaccharides. (A) Total cellular lysates from noninduced (lanes 1 and 3) and tetracycline induced (lanes 2 and 4) HEK293_i-Myc-rLHRvariant cells were precipitated with methanol, subjected to SDS-PAGE under nonreducing (lanes 3 and 4) or reducing (lanes 1 and 2) conditions and analyzed by Western blotting. (B) Membrane vesicles from tetracycline-induced HEK293_i-Myc-rLHR-Flag (lanes 1–4) and HEK293_i-Myc-rLHRvariant (lanes 5–8) cells were subjected to high-salt (lanes 2 and 6), alkaline (lanes 3 and 7), or buffer (lanes 1 and 5) extractions. The extracts supplemented with DDM (lanes 2, 3, 6, and 7) and the DDM-solubilized membranes (lanes 1, 4, 5, and 8) were analyzed by reducing SDS-PAGE and Western blotting. An aliquot of the samples was subjected to Western blot analysis using mouse anti-BiP antibody (bottom panel). (C) Lysates from induced HEK293_i-Myc-rLHR-Flag (lanes 1–3) and HEK293_i-Myc-rLHRvariant (lanes 4–6) cells were subjected to immunoprecipitation with mouse anti-cMyc antibody and immunoprecipitates were incubated for 16 h at 30°C with Endo H (50 mU/ml; lanes 3 and 6), PNGase F (50 U/ml; lanes 2 and 5) or without enzymes (lanes 1 and 4) before reducing SDS-PAGE and Western blotting. The blotted proteins were probed with mouse anti-cMyc antibody (A and B, lanes 1–4, and C) or rabbit anti-receptor antibody directed against the N-terminus of the receptor (B, lanes 5–8). DTT, dithiothreitol. Molecular weight markers used to calibrate the gels are indicated on the right.

Figure 3.

The LHRvariant has difficulties in finding the correct conformation for hormone binding, interacts with calnexin and calreticulin and is a substrate for ERAD. (A) Equal aliquots of lysates from noninduced (lanes 1, 3, 5, 7, 9, 11, and 13) and tetracycline induced (lanes 2, 4, 6, 8, 10, 12, and 14) HEK293_i-Myc-rLHR-Flag and HEK293_i-Myc-rLHRvariant cells and nontransfected HEK293_i cells were subjected to immunoprecipitation and hCG affinity chromatography, as indicated. The purified samples were subjected to reducing SDS-PAGE and Western blotting and probed with mouse anti-cMyc antibody. Lanes 13 and 14 represent a longer exposure of lanes 11 and 12, respectively. (B) Equal aliquots of lysates from noninduced (lanes 1–2 and 5–6) and induced (lanes 3–4 and 7–8) HEK293_i-Myc-rLHRvariant cells were subjected to immunoprecipitation using polyclonal anti-C-terminal calnexin (lanes 1 and 3) or anti-calreticulin (lanes 5 and 7) antibodies or monoclonal anti-cMyc antibody (lanes 2, 4, 6, and 8). The immunoprecipitates were analyzed by reducing SDS-PAGE and Western blotting using anti-N-terminal calnexin (lanes 1–4) or anti-calreticulin (lanes 5–8) antibodies. anti-CnxCT, anti-C-terminal calnexin antibody; anti-Crt, anti-calreticulin antibody. (C) HEK293_i-Myc-rLHRvariant cells were treated with tetracycline for 16 h and lactacystin (10 μM; lane 2) or vehicle (lane 1) was added to the culture medium 6 h before harvesting. Cellular lysates were prepared and methanol-precipitated samples were subjected to reducing SDS-PAGE and Western blotting using mouse anti-cMyc antibody.

Figure 4.

The LHRvariant colocalizes with ER markers calnexin and calreticulin and causes their redistribution. Noninduced (A–C and M–O) and tetracycline induced (D–L and P–R) HEK293_i-Myc-rLHRvariant cells were cultured on coverslips, fixed, and permeabilized before processing for double label indirect immunofluoresence. The ER proteins calreticulin (A–F) and calnexin (G–I) were labeled with rabbit anti-calreticulin and anti-C-terminal calnexin antibodies, respectively, and the ER protein BiP (M–R) and Golgi protein GM130 (J–L) with mouse anti-KDEL and anti-GM130 antibodies, respectively. The LHRvariant was stained in parallel using either mouse (A–I) or rabbit (J–R) anti-cMyc antibody. Secondary antibodies were Alexa 488- or Alexa 568-conjugated goat anti-rabbit and anti-mouse IgG. Colocalization of the variant and the ER markers calnexin and calreticulin in the juxtanuclear region is indicated by arrowheads (F and I). The nuclei were stained with Hoechst in A–F and M–R. Bar, 10 μm.

Figure 5.

The LHRvariant accumulates in the ER and upon proteasomal blockade in the cytosol. Tetracycline induced HEK293_i-Myc-rLHRvariant and HEK293_i-Myc-rLHR-Flag cells were transfected with anti-N-terminal-calnexin (A and G), anti-C-terminal-calnexin (B), anti-cMyc (C and E-F) or anti-FLAG (D) antibodies. After fixation and permeabilization, cells were labeled with secondary antibodies, Alexa 488-conjugated anti-mouse or Alexa 568-conjugated anti-rabbit IgG. The HEK293_i-Myc-rLHR-Flag cells in C and D were treated with brefeldin A (5 μg/ml) during induction to retain the full-length receptor in the ER and proteasomal degradation was inhibited with lactacystin (10 μM) in HEK293_i-Myc-rLHRvariant cells in F and G. Positive cells were counted from 170 to 485 cells/coverslip: 0, 51 ± 7, 0, 64 ± 5, 0, 20 ± 9, 0% (mean ± SEM) for A, B, C, D, E, F, and G, respectively. Bar, 10 μm. anti-CnxCT, anti-C-terminal calnexin antibody; anti-CnxNT, anti-N-terminal calnexin antibody; La, lactacystin.

Figure 6.

The LHRvariant is located in the ER but proteasomal inhibition leads to its appearance in the cytosol. (A–C) Noninduced (A) and tetracycline induced (B and C) HEK293_i-Myc-rLHRvariant cells that were treated or not with lactacystin (10 μM) were fixed, and thin cryosections were labeled with mouse anti-cMyc antibody followed by protein A-gold complex (10 nm). The ER lumen is marked with an asterisk and the arrows indicate gold particles. (D–G) Noninduced (D) and tetracycline induced (E–G) HEK293_i-Myc-rLHRvariant cells were fixed and embedded in plastic, and thin sections were analyzed. Arrows indicate electron-dense material in the compact juxtanuclear tubulo-vesicular body of the induced cells that includes Golgi vesicles and is surrounded by mitochondria. The areas enclosed by squares in E are shown in larger magnification in F and G. Bars, 500 nm (A–C, F and G), 1000 nm (D and E). ER, endoplasmic reticulum; G, Golgi vesicles; La, lactacystin; M, mitochondria; N, nucleus.

Figure 7.

Coexpression with the LHRvariant leads to a decrease in the number of full-length receptors. (A and B) Lysates from noninduced (lane 1) and tetracycline induced (lane 2) HEK293_i-Myc-rLHRvariant cells expressing constitutively the HA-tagged full-length LHR were prepared and receptors were subjected to immunoprecipitation with mouse anti-HA antibody (A) or hCG affinity chromatography (B) and analyzed by reducing SDS-PAGE and Western blotting with anti-HA antibody. (C) HEK293_c cells constitutively expressing the Myc- and Flag-tagged full-length LHR were treated (lane 2) or not (lane 1) with tetracycline for 24 h and lysates were subjected to immunoprecipitation with mouse anti-cMyc antibody. The immunoprecipitates were analyzed by reducing SDS-PAGE and Western blotting using polyclonal anti-cMyc antibody for detection. Panels on the right in A–C show the relative intensities of the mature and immature receptor bands as revealed by densitometric scanning and represent the mean ± SEM of three independent experiments. The intensities in cells not treated with tetracycline were set to 100%. (D) Intact noninduced and induced HEK293_i-Myc-rLHRvariant cells expressing constitutively the HA-tagged full-length LHR were labeled with mouse anti-HA antibody followed by phycoerythrin-conjugated rat anti-mouse antibody and subjected to flow cytometry. The values (mean ± SEM of four independent experiments) indicate the relative mean cell fluorescence compared with the values in noninduced cells that were set to 100%. ***p < 0.0001; **p < 0.009; *p < 0.03; ns, not significant.

Figure 8.

The rLHRvariant expression in HEK293_i cells does not induce unfolded protein response. HEK293_i-Myc-rLHRvariant cells were treated or not with tetracycline and/or tunicamycin, as indicated. The human BiP mRNA expression was analyzed by quantitative PCR. The values indicate the mean relative values (±SEM of three independent experiments) of BiP/18S. The noninduced cells were given a ratio value of 1. **p < 0.01; *p < 0.02; ns, not significant.

Figure 9.

The rLHRvariant redistributes the full-length LHR into a subcompartment of the ER. HEK293_i-Myc-rLHRvariant cells expressing constitutively the HA-rLHR (A–F, M, and N) or transiently the HA-hμOR-Flag (J–L), or alternatively HEK293_i-Myc-rLHR-Flag cells transiently transfected with the HA-rLHRvariant (G–I) or HEK293_c-Myc-rLHR-Flag cells (O and P) were treated or not with tetracycline, as indicated. After permeabilization the HA-tagged rLHR (A–F) and rLHRvariant (G–I) and HA- and Flag-tagged hμOR (J–L) were labeled with mouse anti-HA antibodies and the Myc-tagged rLHRvariant (A–F and J–L) and Myc- and Flag-tagged rLHR (G–I, O, and P) with rabbit anti-cMyc antibody. Alternatively, the cell surface Myc- and Flag-tagged rLHRs (M and N) were labeled before permeabilization with rabbit anti-cMyc antibody. Secondary antibodies were Alexa 488- or Alexa 568-conjugated goat anti-rabbit and anti-mouse IgG. Colocalization of the variant and the full-length receptor in the juxtanuclear region is indicated by arrowheads (F and I). The LHRvariant mediated redistribution of the full-length receptor is clearly evident in the HEK293_i-Myc-rLHR-Flag cells transiently transfected with the HA-rLHRvariant (arrowhead in I), but in nontransfected cells (asterisks in I) the receptor is apparent in a more dispersed reticular pattern. The hμOR did not colocalize significantly with the variant and was transported to the cell surface (arrow in l). The nuclei were stained with Hoechst in O and P. Bar, 10 μm.