A, Schematic representation of the exon and putative protein organization of the LGR7 splice variants cloned from the placental basal plate. The cDNA of the full-length WT-LGR7 spliced from 18 exons is shown for comparison (15). Exons are represented as boxes; exon 1 contains the signal sequence (black box), the LDL-A module encoded by exon 2 (white box), the 10 LRRs encoded from exons 5–14 (black boxes), and the TM domain encoded by the sequence from exons 15–18 (striped boxes). In all three splice variants, deletion of exons leads to shifts in the open reading frame and to premature termination of their putative polypeptides. Splice variants LGR7-D and LGR7-F use cryptic splice acceptor and splice donor sites. Shifts in the open reading frames resulted in novel amino acid sequences after the alternative splicing sites, not present in the full-length receptor. This alternative splicing resulted in proteins containing different lengths of the extracellular region of the LGR7. B, Expression of mRNAs for the splice variants and WT-LGR7 in amnion and decidua from monozygotic twin pregnancies (n = 3). Representative mRNAs from PCR analysis of one tissue are shown. The mRNA analysis of the splice variants was performed by nested PCR, WT-LGR7 and GAPDH transcripts by regular PCR. The oligonucleotide sequences of the primers are shown in Table 1. The amnion was isolated from the septum of the fetal membranes separating twin gestations that contained no chorion because these were monozygotic twins and decidua from the placental basal plates. Total mRNA from uterus was obtained commercially. Positive controls contained a reaction mix that included plasmids containing the cDNA sequences of the splice variants or WT-LGR7 as a template. No PCR products were detected in controls containing water instead of the cDNAs, and the housekeeping gene GAPDH was used. The sizes of PCR products were as follows: splice variant C, 268 bp; splice variant D, 240 bp; splice variant F, 258 bp; WT-LGR7, 244 bp; and GAPDH, 140 bp.

Detection of the splice variant proteins expressed in transfected HEK293 cells by Western blot. A, Total membrane and cytosol fractions from HEK293 cells transfected with each splice variant with the HA sequence inserted after the signal peptide for each cDNA. Samples (5 μg) of total membrane proteins and cytosol fractions were separated on 10% SDS-PAGE, immunoblotted, and incubated with an antibody to HA. All three splice variants were localized in the membrane fraction and none detected in the cytosol. No signal was detected in membrane fractions from cells transfected with an empty vector (control). B, Splice variant LGR7-D has a secreted protein form. Lysates and medium from HEK293 cells transfected with each splice variant cDNA with an HA tag were collected and subjected to immunoprecipitation with an anti-HA-agarose. Immunoprecipitates were separated in 10% gels and analyzed on Western blotting with an HA antibody. All three splice variants were detected in the cell lysates, and only splice variant LGR7-D was detected in medium and had a higher molecular weight compared with its intracellular form.

Secreted LGR7-D is delivered to the cell surface of transfected cells. A, HEK293 cells transfected with WT-LGR7, LGR7-C, LGR7-D, or LGR7-F bearing the HA epitope at their N termini as well as LGR7-HA bearing the HA epitope at its C terminus (control) were assayed by indirect ELISA using an antibody to the HA tag to show cell surface expression. Relative cell surface expression (cell surface expression to total expression) is shown as percentage of relative cell surface expression of the HA-WT-LGR7. ***, P < 0.001 compared with HA-WT-LGR7 by Student’s paired t test. B, Confocal microscopic images of nonpermeabilized (upper panels) and permeabilized (lower panels) HEK293 cells transfected with WT-LGR7, LGR7-C, LGR7-D, and LGR7-F bearing the HA tag at their N termini. The cells were stained with primary antibody to HA and with mouse AlexaFluor 488-conjugated secondary antibody, showing cell surface localization of the WT-LGR7 and LGR7-D in nonpermeabilized cells and the intracellular distribution of all constructs in the permeabilized cells. C, Expression analysis of an LGR7-D glycosylation mutant (N36Q-D) by cell surface ELISA. HEK293 cells transfected with WT-LGR7-D and N36Q-D bearing the HA tag at their N termini were assayed by cell surface ELISA. Cell surface expression was expressed as percentage of WT-LGR7-D and shows that cell surface expression of N36Q-D was reduced to 45.4 ± 1.8% compared with WT-LGR7-D. ***, P < 0.001 using Student’s paired t test.

Effects of the LGR7 splice variants on the expression of the WT-LGR7. A, Effects of the splice variants on the cell surface delivery of WT-LGR7 by ELISA. HEK293 cells were cotransfected with HA-WT-LGR7 and either one splice variant construct or an empty vector (control) and assayed with the cell surface ELISA. Transfections were performed with different ratios of splice variants or empty vector to HA-WT-LGR7. Cell surface expression is presented as percentage of the HA-WT-LGR7 expression in the presence of the empty vector. ***, P < 0.001; ** P < 0.01 compared with HA-WT-LGR7 coexpressed with an empty vector by Student’s paired t test. B, The presence of a splice variant leads to decreased maturation of the full-length receptor. Lysates from cells coexpressing the HA-WT-LGR7 and one of the splice variants or an empty vector (control) were immunoprecipitated (IP) with HA-agarose and analyzed by 7.5% SDS-PAGE and Western blotting with an HA antibody (upper panel). The membrane preparations from cells transfected with HA-WT-LGR7 were also detected by Western blotting (WB). Two expressed molecular weight forms of HA-WT-LGR7 (95-kDa mature and 80-kDa precursor) were detected (upper panel). The relative intensities of the mature form were determined by densitometric analysis (mean ± sem of three independent experiments). Data are expressed as percentage intensity in cells coexpressing HA-WT-LGR7 and empty vector (lower panel). ***, P < 0.001 by Student’s paired t test analysis. C, The splice variants did not induce an UPR. Cells respond to ER stress by increasing transcription of genes encoding resident chaperones for facilitation of protein folding. mRNA from untreated HEK293 cells, HEK293 cells treated with tunicamycin (Tunic., an UPR-inducing reagent, 5 μg/ml) for 24 h (positive control for BiP induction), and HEK293 cells stably transfected with WT-LGR7, LGR7-C, LGR7-D, or LGR7-F were isolated and the BiP mRNA expression analyzed by quantitative real-time PCR. Data are shown as mean relative values of BiP/18S ± sem of three independent experiments. The mean result from untreated HEK293 cells was arbitrarily assigned a value of 1. No significant changes were observed in BiP expression in HEK293 cells compared with HEK293 cells with stable expression of each splice variant or WT-LGR7. In contrast, tunicamycin treatment led to a significant increase in BiP mRNA levels. **, P < 0.01 compared with untreated HEK293 cells; ns, not significant. D, The splice variants stably interact with WT-LGR7. Lysates from untransfected cells (HEK U/T, lane 1), cells cotransfected with HA-WT-LGR7 and one splice variant (lanes 2–4), and cells cotransfected with HA-WT-LGR7 and an empty GFP vector (lane 5) were immunoprecipitated with HA-agarose. The immunoprecipitates (IP) were analyzed by 7.5% SDS-PAGE and Western blotting (WB) using a rabbit antibody to GFP. Splice variants were detected in the immunoprecipitates from the cotransfected cells (lanes 2–4) but not from the untransfected controls or cells cotransfected with HA-WT-LGR7 and an empty vector. This shows the direct interactions between the splice variants and the WT-LGR7. E, The splice variants colocalize with WT-LGR7. Cells cotransfected with HA-WT-LGR7 and an empty GFP vector (a–c) or cotransfected with HA-WT-LGR7 and a splice variant tagged with GFP (splice variant C, d–f; splice variant D, g–i; and splice variant F, j–l) were fixed, permeabilized, and stained for the HA epitope with a primary mouse HA antibody and secondary mouse AlexaFluor 546 antibody. Colocalization of each variant and HA-WT-LGR7 is shown on the overlays (f, i, and l). No colocalization was detected in cells coexpressing HA-WT-LGR7 and the empty GFP vector (c).

Constitutive dimerization of the LGR7s occurs in the ER and PM. Total membrane preparations from HEK293 cells cotransfected with LGR7-Rluc and LGR7-YFP or LGR7-Rluc and WT-LGR7 were fractionated on a sucrose gradient and fractions subjected to BRET measurements (upper panel). The individual fractions were resolved on 7.5% SDS-PAGE and Western blotting performed using antibody markers for PM (Na/K ATPase) and ER (calnexin). The data shown are representative of two independent experiments.

Comparative mRNA expression levels for the WT-LGR7 splice variants LGR7-C, LGR7-D, and LGR7-F in separated decidua, chorion, and amnion from singleton term pregnancies both before or no labor (NL) and after spontaneous labor (SL) (n = 5). Relative expression levels were determined by real-time quantitative RT-PCR using primers designed to amplify WT-LGR7 and each variant specifically (Table 1). Results are expressed as means ± sem of relative mRNA normalized to the levels of 18S RNA in each sample. The mean results of NL decidua for WT-LGR7, LGR7-C, and LGR7-D and the mean result of NL chorion for LGR7-F were arbitrarily assigned to value 1. No variant expression was detected (n/d) in the amnion, and LGR7-F was also undetectable in the decidua. *, P < 0.05.

Localization of the splice variants to the ER. A, Lysates of cells transfected with LGR7-C, LGR7-D, or LGR7-F constructs containing the HA tag or medium from cells transfected with LGR7-D with the HA tag were immunoprecipitated with an antibody to HA-agarose. Immunoprecipitates were incubated with Endo H, PNGase F, or without enzymes, separated on 10% gels, blotted, and probed with the HA antibody. B, The splice variants colocalize with ER markers calnexin and calreticulin. HEK293 cells transfected with each splice variant with an HA-tag were fixed, permeabilized, and double labeled for confocal microscopy. The ER resident proteins were labeled with rabbit antisera to calnexin (left panel) or calreticulin (right panel). The splice variants LGR7-C (a–c), LGR7-D (d–f), and LGR7-F (g–i) were immunostained in parallel using a mouse antibody to HA. Secondary antibodies were AlexaFluor 488 (mouse) or AlexaFluor 546 (rabbit). Overlay pictures (c, f, and i) show strong colocalization of the three splice variants with calnexin and calreticulin, respectively, and their localization in the ER.

Functional characterization of the LGR7 splice variants. A, Relaxin caused a dose-dependent stimulation of cAMP production from HEK293 cells transfected with WT-LGR7, but cells expressing LGR7-C, LGR7-D, or LGR7-F failed to show any effects. cAMP accumulation is expressed as percentage of the maximum relaxin response of WT-LGR7. B, Effects of the LGR7 splice variants on the function of the WT-LGR7. Relaxin stimulated the dose-dependent production of cAMP from cells cotransfected with WT-LGR7 and an empty vector (control, dashed line). Efficacy of relaxin-stimulated cAMP production of the WT-LGR7 was significantly decreased (P < 0.05) in the presence of each splice variant (solid lines) compared with WT-LGR7 with the empty vector (dashed line). Accumulation of cAMP was expressed as percentage of the maximum relaxin response in the presence of WT-LGR7 and empty vector.

Homodimerization of the LGR7. A, The WT-LGR7 was tagged at its C terminus with Renilla luciferase (LGR7-Rluc) or YFP (LGR7-YFP). Production of cAMP was assayed in cells transfected with WT-LGR7, LGR7-Rluc, and LGR7-YFP after treatment with different doses of relaxin (0.1, 1, and 10 nm). There were no significant differences in cAMP production in cells expressing LGR7-Rluc or LGR7-YFP compared with WT-LGR7. Thus, fusion of LGR7 to Rluc or YFP had no effect on the signaling of LGR7. B, Homodimerization of LGR7 assessed by BRET saturation assay. HEK293 cells were cotransfected with a constant amount of LGR7-Rluc and increasing DNA concentrations of LGR7-YFP. Data are shown as net BRET signals plotted over the relative expression levels (total fluorescence over luminescence, YFP/Rluc) and results expressed as the mean ± sem of three independent experiments. The curve was fitted using nonlinear regression equation assuming a single binding site (GraphPad Prism). C, BRET competition assays. HEK293 cells coexpressing a constant DNA level of LGR7-Rluc and LGR7-YFP constructs (concentrations determined to give a signal close to the BRET₅₀) in the presence of an empty vector or untagged LGR7. The maximal net BRET measured in the presence of an empty vector was set at 100%. Data are expressed as a mean ± sem of three experiments. The BRET signal obtained in the presence of untagged LGR7 was significantly lower compared with the BRET signal measured in cells coexpressing LGR7-Rluc, LGR7-YFP, and an empty vector. ***, P < 0.001 compared with net BRET signal in the presence of an empty vector.

Effects of LGR7 splice variants on dimerization of the WT-LGR7. BRET saturation assays for LGR7 dimerization were carried out in the presence of each splice variant. HEK293 cells were cotransfected with a constant amount of LGR7-Rluc and increasing DNA concentrations of LGR7-YFP in the presence of an empty vector (control) or the splice variants (LGR7-C, upper panel; LGR7-D, middle panel; and LGR7-F, lower panel). Data are shown as net BRET signals plotted over the relative expression levels (total fluorescence over luminescence; YFP/Rluc). The curves (solid line, in the presence of empty vector; dashed line, in the presence of splice variant) were fitted using nonlinear regression equation assuming a single binding site (GraphPad Prism). The titration curves were shifted to the right when splice variants were present, showing that each splice variant inhibited formation of WT-LGR7 dimers.