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• W2016088181 abstract "Gonadotropin-releasing hormone (GnRH) receptor mutants from patients with hypogonadotropic hypogonadism are frequently misrouted proteins that exert a dominant-negative (DN) effect on human (h) wild-type (WT) receptor, due to oligomerization and retention in the endoplasmic reticulum. Pharmacologic chaperones restore correct folding, rescuing mutants and WT receptor from this oligomer. Rat WT retains the ability to oligomerize (since human and mouse mutants exert a DN effect on rat (r) WT sequence) but, unlike human or mouse, escapes the DN effect of GnRH receptor (Gn-RHR) mutants because rGnRHR mutants route to the plasma membrane with higher efficiency than mouse or human mutants. These distinct behaviors of mouse and rat GnRHRs (distinguished by only four semi- or non-conservative amino acid differences) led us to assess the role of each amino acid. The difference in both routing and the DN effect appears mediated primarily by Ser216 in the rGnRHR. The homologous amino acid in the hGn-RHR is also Ser and is compensated for by the primate-unique insertion of Lys191 that, alone, dramatically decreases routing of the receptor. These studies establish the relation between the DN effect and altered receptor trafficking and explain why hGnRHR is more susceptible to defective trafficking by disease-related point mutations than rodent counterparts. Gonadotropin-releasing hormone (GnRH) receptor mutants from patients with hypogonadotropic hypogonadism are frequently misrouted proteins that exert a dominant-negative (DN) effect on human (h) wild-type (WT) receptor, due to oligomerization and retention in the endoplasmic reticulum. Pharmacologic chaperones restore correct folding, rescuing mutants and WT receptor from this oligomer. Rat WT retains the ability to oligomerize (since human and mouse mutants exert a DN effect on rat (r) WT sequence) but, unlike human or mouse, escapes the DN effect of GnRH receptor (Gn-RHR) mutants because rGnRHR mutants route to the plasma membrane with higher efficiency than mouse or human mutants. These distinct behaviors of mouse and rat GnRHRs (distinguished by only four semi- or non-conservative amino acid differences) led us to assess the role of each amino acid. The difference in both routing and the DN effect appears mediated primarily by Ser216 in the rGnRHR. The homologous amino acid in the hGn-RHR is also Ser and is compensated for by the primate-unique insertion of Lys191 that, alone, dramatically decreases routing of the receptor. These studies establish the relation between the DN effect and altered receptor trafficking and explain why hGnRHR is more susceptible to defective trafficking by disease-related point mutations than rodent counterparts. The gonadotropin-releasing hormone (GnRH) 1The abbreviations used are: GnRH, gonadotropin-releasing hormone; GnRHR, gonadotropin-releasing hormone receptor; DN, dominant-negative; HH, hypogonadotropic hypogonadism; WT, wild-type; h, human; m, mouse; r, rat; DMEM, Dulbecco's modified Eagle's medium; Buserelin, d-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH; IN3, (2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine; IP, inositol phosphate. receptor (Gn-RHR) is a heptahelical G protein-coupled receptor found in the plasma membrane of pituitary gonadotropes (1Ulloa-Aguirre A. Conn P.M. Handbook of Physiology-Endocrinology. 7. Oxford University Press, New York1998: 87-124Google Scholar, 2Millar R.P. Lu Z-L. Pawson A.J. Flanagan C.A. Morgan K. Maudsley S.R. Endocr. Rev. 2004; 25: 235-275Crossref PubMed Scopus (631) Google Scholar). GnRHR mutants isolated from patients with hypogonadotropic hypogonadism (HH) are frequently misfolded (and consequently mislocalized) proteins, restored to function by pharmacological chaperones (“pharmacoperones,” low molecular weight peptidomimetics of several chemical classes that bind the GnRHR) (3Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar, 4Maya-Núñez G. Janovick J.A. Ulloa-Aguirre A. Söderlund D. Conn P.M. Méndez J.P. J. Clin. Endocrinol. Metab. 2002; 87: 2144-2149Crossref PubMed Scopus (66) Google Scholar, 5Leaños-Miranda A. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 4825-4828Crossref PubMed Scopus (109) Google Scholar, 6Ulloa-Aguirre A. Janovick J.A. Leaños-Miranda A. Conn P.M. Expert Opin. Ther. Targets. 2003; 7: 175-185Crossref PubMed Scopus (31) Google Scholar, 7Conn P.M. Leaños-Miranda A. Janovick J.A. Mol. Interv. 2002; 5: 308-316Crossref Scopus (59) Google Scholar). These serve as templates (8Ulloa-Aguirre A. Janovick J.A. Brothers S.P. Conn P.M. Traffic. 2004; 5: 821-837Crossref PubMed Scopus (240) Google Scholar, 9Janovick J.A. Goulet M. Bush E. Greer J. Wettlaufer D.G. Conn P.M. J. Pharmacol. Exp. Ther. 2003; 305: 608-614Crossref PubMed Scopus (120) Google Scholar) that reshape mutants so that they pass the quality control apparatus of the cell. As a result, originally misfolded mutants are then correctly routed to the plasma membrane and become functional (3Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar, 4Maya-Núñez G. Janovick J.A. Ulloa-Aguirre A. Söderlund D. Conn P.M. Méndez J.P. J. Clin. Endocrinol. Metab. 2002; 87: 2144-2149Crossref PubMed Scopus (66) Google Scholar, 5Leaños-Miranda A. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 4825-4828Crossref PubMed Scopus (109) Google Scholar, 6Ulloa-Aguirre A. Janovick J.A. Leaños-Miranda A. Conn P.M. Expert Opin. Ther. Targets. 2003; 7: 175-185Crossref PubMed Scopus (31) Google Scholar, 7Conn P.M. Leaños-Miranda A. Janovick J.A. Mol. Interv. 2002; 5: 308-316Crossref Scopus (59) Google Scholar, 8Ulloa-Aguirre A. Janovick J.A. Brothers S.P. Conn P.M. Traffic. 2004; 5: 821-837Crossref PubMed Scopus (240) Google Scholar, 9Janovick J.A. Goulet M. Bush E. Greer J. Wettlaufer D.G. Conn P.M. J. Pharmacol. Exp. Ther. 2003; 305: 608-614Crossref PubMed Scopus (120) Google Scholar, 10Ulloa-Aguirre A. Janovick J.A. Leaños-Miranda A. Conn P.M. Hum. Reprod. Update. 2004; 10: 177-192Crossref PubMed Scopus (43) Google Scholar, 11Castro-Fernandez C. Maya-Nunez G. Conn P.M. Endocr. Rev. 2005; 26: 479-503Crossref PubMed Scopus (69) Google Scholar). GnRHR mutants (12Karges B. Karges W. de Roux N. Hum. Reprod. Update. 2003; 6: 523-530Crossref Scopus (51) Google Scholar, 13de Roux N. Young J. Misrahi M. Genet R. Chanson P. Schaison G. Milgrom E. N. Engl. J. 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Metab. 1999; 84: 990-996Crossref PubMed Google Scholar, 19Kottler M.L. Chauvin S. Lahlou N. Harris C.E. Johnston C.J. Lagarde J.P. Bouchard P. Farid N.R. Counis R. J. Clin. Endocrinol. Metab. 2000; 85: 3002-3008Crossref PubMed Scopus (71) Google Scholar, 20Costa E.M.F. Bédécarrats G.Y. Mendonca B.B. Arnhold I.J.P. Kaiser U.B. Latronico A.C. J. Clin. Endocrinol. Metab. 2001; 86: 2680-2686Crossref PubMed Scopus (100) Google Scholar, 21Karges B. Karges W. Mine M. Ludwig L. Kühne R. Milgrom M. de Roux N. J. Clin. Endocrinol Metab. 2003; 88: 1873-1879Crossref PubMed Scopus (55) Google Scholar, 22Kottler M.L. Counis R. Bouchard P. Arch. Med. Res. 1999; 30: 481-485Crossref PubMed Scopus (37) Google Scholar, 23Bédécarrats G.Y. Linher K.D. Kaiser U.B. J. Clin. Endocrinol. Metab. 2003; 88: 834-843Crossref PubMed Scopus (46) Google Scholar) obtained from such patients also inhibit ligand binding and ligand-activated second messenger production by wild-type (WT) receptors when co-expressed in vitro, a dominant-negative (DN) effect (24Leaños-Miranda A. Ulloa-Aguirre A. Ji T.H. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2003; 88: 3360-3367Crossref PubMed Scopus (63) Google Scholar). The cellular etiology of this effect is a stable physical interaction between the mutant and wild-type GnRHR, producing an oligomer that is recognized by the cellular quality control apparatus as a defective complex (25Brothers S.P. Cornea A. Janovick J.A. Conn P.M. Mol. Endocrinol. 2004; 18: 1787-1797Crossref PubMed Scopus (94) Google Scholar). This oligomer is retained by the endoplasmic reticulum then presumably degraded (or recycled) (11Castro-Fernandez C. Maya-Nunez G. Conn P.M. Endocr. Rev. 2005; 26: 479-503Crossref PubMed Scopus (69) Google Scholar, 25Brothers S.P. Cornea A. Janovick J.A. Conn P.M. Mol. Endocrinol. 2004; 18: 1787-1797Crossref PubMed Scopus (94) Google Scholar); plasma membrane expression of the WT receptor is thereby diminished. Not surprisingly, pharmacoperones also reverse the DN effect (24Leaños-Miranda A. Ulloa-Aguirre A. Ji T.H. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2003; 88: 3360-3367Crossref PubMed Scopus (63) Google Scholar, 25Brothers S.P. Cornea A. Janovick J.A. Conn P.M. Mol. Endocrinol. 2004; 18: 1787-1797Crossref PubMed Scopus (94) Google Scholar) of this mutant on the wild-type receptor, since the rescued oligomer of the WT and mutant pass the cellular quality control apparatus. In the present study, unexpected and dramatic differences in both the DN effect and plasma membrane routing of rat and mouse GnRHR mutants were observed; rat, but not mouse, mutants failed to show a DN effect when co-expressed with WT receptor. This was surprising in light of the modest differences (four semi- or non-conservative changes) between the rat and mouse sequences. To determine the residue(s) responsible for the loss of dominant negativity by the rWT GnRHR, mouse, and rat homologs of the DN human mutant Glu90 → Lys (E90K) were prepared along with WT and other HH mutants in which combinations of the four semi- or non-conservative changes were made (mouseXrat: P11Q, I24T, I160T, and G216S, where X is the amino acid sequence position). The mutants were shown to be misrouted proteins but when rescued with pharmacoperones became fully functional “receptors” able to bind ligand and couple to the effector system. Additionally, the role of a primate-specific amino acid, Lys191, that is inserted in the human receptor sequence (328 amino acids) (20Costa E.M.F. Bédécarrats G.Y. Mendonca B.B. Arnhold I.J.P. Kaiser U.B. Latronico A.C. J. Clin. Endocrinol. Metab. 2001; 86: 2680-2686Crossref PubMed Scopus (100) Google Scholar) was examined. The presence of Lys191 in the human GnRHR is associated with a decrease in the proportion of plasma membrane localized receptors. Like defective routing of mutants, this effect is superseded by pharmacoperones that rescue the mutant GnRHRs. Rodent GnRHRs (327 amino acids), which are normally transferred to the plasma membrane with higher efficiency than primate GnRHRs, lack this amino acid insertion. This observation suggests that the human sequence is normally trafficked to the plasma membrane with less than 100% efficiency. Removal of Lys191 from the human GnRHR(E90K) sequence rescues this mutant and increases the efficiency of hWT GnRHR routing the plasma membrane. The role of this primate-specific modification was examined with regard to the amino acid that governs the highly efficient transfer of rat WT GnRHR to the plasma membrane and loss of the DN effect of its mutants. Material—pcDNA3.1+ (Invitrogen), the GnRH analog, d-tert-butyl-Ser6-des-Gly10-Pro9-ethylamide-GnRH (Buserelin; Hoechst-Roussel Pharmaceuticals, Somerville, NJ), (2S)-2-[5-[2-(2-azabicyclo[2.2.2]oct-2-yl)-1,1-dimethyl-2-oxoethyl]-2-(3,5-dimethylphenyl)-1H-indol-3-yl]-N-(2-pyridin-4-ylethyl)propan-1-amine (IN3; Merck), myo-[2-3H]inositol (New England Nuclear, Boston, MA; NET-114A), DMEM, Opti-MEM, Lipofectamine, phosphate-buffered saline (Invitrogen), competent cells (Promega, Madison, WI), and endofree plasmid maxi-prep kits (Qiagen, Valencia, CA) were obtained as indicated. Other reagents were obtained from commercial sources. Rodent and human WT and mutant GnRHR cDNA were prepared as reported (3Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar, 26Horton R.M. Ho S.N. Pullen J.K. Hunt H.D. Cai Z. Pease L.R. Methods Enzymol. 1993; 217: 270-279Crossref PubMed Scopus (432) Google Scholar). Transient Transfection and Co-transfection—Cells were cultured and plated in growth medium (DMEM, 10% fetal calf serum, 20 μg/ml gentamicin), and growth conditions were 37 °C and 5% CO2 in a humidified atmosphere; all medium added to the cells was warmed to 37 °C prior to adding to the cells, unless otherwise noted. For transfection of WT or mutant receptors into COS-7 cells, 5 × 104 cells were plated in 0.25 ml of growth medium in 48-well Costar cell culture plates. Twenty-four h after plating, the cells were washed once with 0.5 ml of Opti-MEM and then transfected with 100 ng of total cDNA (pcDNA3.1+ without insert “empty vector” was included to bring the total cDNA to 100 ng/well, unless otherwise indicated) and 1 μl of Lipofectamine in 0.125 ml of Opti-MEM (room temperature), according to manufacture's instructions. For co-transfection experiments the cells were co-transfected with WT GnRHR (5 ng/well) and empty vector or mutant GnRHR (95 ng/well). Five h after transfection, 0.125 ml of DMEM with 20% fetal calf serum and 20 μg/ml gentamicin was added to the wells. Twenty-three h after transfection, the medium was removed and replaced with 0.25 ml of fresh growth medium. Where indicated, 1 μg/ml IN3 in 0.1% final dimethyl sulfoxide (vehicle) was added in respective media to the cells and then removed 18 h before agonist treatment, as described elsewhere (3Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar, 5Leaños-Miranda A. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 4825-4828Crossref PubMed Scopus (109) Google Scholar, 24Leaños-Miranda A. Ulloa-Aguirre A. Ji T.H. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2003; 88: 3360-3367Crossref PubMed Scopus (63) Google Scholar). Data are presented as the means ± S.E., calculated from at least three independent experiments, each performed in replicates of six. Inositol Phosphate (IP) Assays—Twenty-seven h after transfection, cells were washed twice with 0.5 ml of DMEM containing 0.1% bovine serum albumin and 20 μg/ml gentamicin and then “preloaded” for 18 h with 0.25 ml of 4 μCi/ml myo-[2-3H]inositol in DMEM (prepared without inositol). After preloading, cells were washed twice with 0.3 ml of DMEM containing 5 mm LiCl (without inositol), then treated for 2 h in 0.25 ml of a concentration of Buserelin (10–7 m Buserelin, unless otherwise indicated) in the same medium (LiCl prevents IP degradation). The media were removed, and the cells were frozen and thawed in the presence of 0.5 ml of 0.1 m formic acid (to rupture cells), and total IPs were determined as described previously (30Huckle W. Conn P.M. Methods Enzymol. 1987; 141: 149-155Crossref PubMed Scopus (55) Google Scholar) by liquid scintillation counting. Data were normalized to correct for differences in counting efficiency and for differences in specific activity of tritiated inositol lots. Statistics—The Student's paired t test (SigmaStat 3.1) was used to determine significance, with p values < 0.05 considered significant. One-way analysis of variance was used to determine interexperimental variance between data sets. Structural Features of the GnRHR—Fig. 1A is a graphic of the rat WT GnRHR sequence, indicating the four positions of semi- or non-conservative mutations between the rat and mouse sequence (shaded boxes: mouseXrat: P11Q, I24T, I160T, and G216S, where “X” is the amino acid position). The amino acid at each of the four positions in the human sequence is also shown for comparison (shaded boxes). Conservative substitutions compared with the mouse sequence are shown in the small unshaded boxes. The human WT GnRHR also contains an “extra” amino acid, Lys191 (small circle, site of insertion shown by a black arrow); this amino acid is absent in all pre-primate species sequenced to date. The site of amino acid E90K, a mutant examined in this study, is shown (small circle, location shown with a black arrow) in the second transmembrane portion of the molecule. The comparative sequences for the fifth transmembrane domain of the three species are shown. Substantial Evolutionary Homology between the Rodent and Human GnRHR—Fig. 1B (top right) also shows a phylogram tree constructed from the reported sequences (2Millar R.P. Lu Z-L. Pawson A.J. Flanagan C.A. Morgan K. Maudsley S.R. Endocr. Rev. 2004; 25: 235-275Crossref PubMed Scopus (631) Google Scholar) of mammalian GnRHRs and specifically the relationship between rat, mouse, and human receptors studied in the present work, along with others. The mGnRHR sequence is slightly closer to the human sequence (89% homology) than the rGnRHR (88% homology), indicated by the shorter line length. The two rodent GnRHRs are 96% homologous to each other. GnRHR Mutants Exert a DN Effect on the Wild-type Gn-RHR—Fig. 2A is an agonist dose response of IP production in cells transfected with 95 ng of human WT GnRHR. The absence of Buserelin produces a response only slightly above cells that did not contain GnRHR (note broken x axis in image). Cells lacking transfected GnRHR or mutant (i.e. cells containing empty vector only) do not respond to Buserelin (see legends to Figs. 6, 7, 8, 9, 10 for vector-only control values). 10–7 m of the GnRH agonist, Buserelin, produces the maximal response and for this reason that amount was used in subsequent studies. Fig. 2B shows the DN effect of the hGnRHR(E90K) mutant on function of the human WT GnRHR. Although the DN effect of human E90K on human WT is significant (p < 0.05) at a ratio of 3:1 (mutant:WT), a 19:1 ratio was used in the present study for a more pronounced effect. In vivo, each cell in a human heterozygote (mutant:WT) would likely express those genes equally, a circumstance that may not occur when equal amounts of vectors are transfected into cells in vitro. Furthermore, since heterozygotic patients expressing highly DN mutants would likely be infertile, such mutations would have been selected against and those that appear in the population would be among the least severe in this regard. Accordingly, higher ratios were used in the present study.Fig. 6Pharmacological rescue of mouse GnRHR(E90K)-derived mutants. Transfected COS-7 cells (95 ng of mutant or WT with 5 ng of vector) were stimulated with 10–7 m Buserelin, and IP production was measured. Data represent the mean (n = 6) cpm ± S.E. of a representative experiment. “Vehicle” data (dimethyl sulfoxide (DMSO)) shows IP production in the absence of the pharmacoperone. When the rescue pharmacoperone IN3 was present it was added at 1 μg/ml, shown previously (3Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar) to result in optimum rescue. Transfection of 100 ng of empty vector (data not shown) produced a response in the absence of IN3 of 66.8 ± 2.1 cpm and 62.6 ± 2.1 cpm when IN3 was present.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 7Co-transfection of rat, mouse, and human WT GnRH receptor with double, triple, and quadruple mouse GnRHR(E90K) mutants. Rat, mouse, and human WT GnRHR sequences (listed above graphs) contain the naturally occurring amino acid 216 (Ser216 for rat and human and Gly216 for mouse). The dashed lines indicate the IP production level of the three species WT GnRHR when co-transfected with 95 ng of vector. The substitutions (listed above the top image in the legend) at position 216 were made to the rodent sequences co-transfected with WT GnRHR sequences. When comparing E90K mutant co-transfections note that the rat GnRHR(E90K) contains the Ser216 residue. Co-transfected COS-7 cells (5 ng of WT with 95 ng of vector or mutant DNA) were stimulated with 10–7 m Buserelin, and IP production was measured. Data represent the mean (n = 6) cpm ± S.E. of a representative experiment. 100 ng of empty vector (data not shown) produced 81.4 + 2.9 cpm. *a, p < 0.05 when compared with co-transfection of rWT GnRHR and vector. *b, p < 0.05 when compared with co-transfection of mWT GnRHR and vector.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 8Effect of Lys191 on plasma membrane expression and the DN effect of the rodent GnRHR(+Lys191) and human GnRHR(–Lys191) 216/217 double mutants. A, mutant and WT Gn-RHR sequences were transfected in COS-7 cells (95 ng of WT with 5 ng of vector DNA) and stimulated with 10–7 m Buserelin, and IP production was measured. Data represent the mean (n = 6) cpm ± S.E. of a representative experiment. The indicated sequences were expressed in COS-7 to assess the effect of Lys191 independently and in combination with glycine or serine at position 216 in the GnRH receptor sequence. B–D, the DN action of the mutant sequences from A (95 ng of DNA) on WT sequences (listed above graph in a box, 5 ng of DNA) was assessed as described under “Experimental Procedures.” WT GnRHR sequences were co-transfected in COS-7 cells (5 ng of WT with 95 ng of mutant or vector DNA) and stimulated with 10–7 m Buserelin, and IP production was measured. Data are presented as the mean (n = 6) cpm ± S.E. of a representative experiment. For comparison, 5 ng of rat, mouse, or human WT with 95 ng of empty vector is shown on the left of B–D. 100 ng of empty vector (data not shown) produced 109.0 ± 2.0 cpm (A) and 98.3 ± 3.3 cpm (B–D).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 9Mutations in the human GnRHR from patients with hypogonadotropic hypogonadism were reconstructed in the mouse and rat receptor sequences. Mutant and WT GnRHR sequences were transfected in COS-7 cells (95 ng of WT with 5 ng of vector DNA) and stimulated with 10–7 m Buserelin, and IP production was measured. Data represent the mean (n = 6) cpm ± S.E. of one representative experiment. Vehicle only (dimethyl sulfoxide (DMSO)) data show IP production in the absence of the pharmacoperone. When the rescue pharmacoperone IN3 was present, it was added at 1 μg/ml, shown previously (3Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar) to result in optimum rescue. 100-ng transfection of empty vector (data not shown) produced a response, in the absence of IN3, of 72.3 + 2.9 cpm, and 69.6 + 1.7 cpm when IN3 was present.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 10The four semi- and non-conservative rodent differences were constructed in the mWT GnRH receptor sequence. WT GnRHR sequences were transfected in COS-7 cells (95 ng of WT with 5 ng of vector DNA) and stimulated with 10–7 m Buserelin, and IP production was measured. Data represent the mean (n = 6) cpm ± S.E. of one representative experiment. A, expression of mouse WT GnRHR-derived mutants, whereas the bottom images (B–D) show co-transfection with indicated WT GnRHR (listed above graph). B, WT GnRHR sequences were co-transfected in COS-7 cells (5 ng of WT with 95 ng of mutant or vector DNA) and stimulated with 10–7 m Buserelin, and IP production was measured. Data are presented as the mean (n = 6) cpm ± S.E. of a representative experiment. The dashed lines indicate IP production of WT GnRHR co-transfected with vector. Vector alone (data not shown) produced IP responses of 71.6 ± 0.9 cpm (A) and 81.4 ± 2.9 cpm (B–D).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Lys191, a Primate-specific Amino Acid Addition, Decreases Expression of the Human Sequence and Rescues Expression of the E90K Mutant—Fig. 3 shows IP production data for rat, mouse, and human WT and the E90K mutant of the GnRH receptor. Each sequence was expressed as described under “Experimental Procedures” in COS-7 cells, and IP production was used as an indication of receptor-effector coupling. We have shown previously that IP production accurately reflects plasma membrane expression of these moieties (25Brothers S.P. Cornea A. Janovick J.A. Conn P.M. Mol. Endocrinol. 2004; 18: 1787-1797Crossref PubMed Scopus (94) Google Scholar). Fig. 3 also shows the effect of deleting the Lys191 residue from the human (WT or mutant E90K) GnRHR sequence. In addition, effects of the insertion of this amino acid into rat and mouse WT and E90K-derived sequences were examined. Removal of Lys191 from the human sequence increases expression of both the WT and mutant E90K forms of the receptor, since this amino acid normally precludes complete expression of the receptor at the plasma membrane. Interestingly, mouse and rat WT are apparently not affected by the Lys191 insertion. In both rodent sequences, expression of E90K mutants was reduced by the Lys191 insertion. The rat GnRHR(E90K) mutant expresses 15.06 + 0.32% compared with the WT, followed by very modest expression of the mouse GnRHR(E90K) mutant (3.59 + 0.20%) compared with the WT. The human E90K mutant is indistinguishable from a “vector only” control (vector only data in figure legend). Interspecific Specificity of the DN Effect—The species specificity of the rat, mouse, and human GnRHRs modified at E90K to exert a DN effect on the WT receptor from each of the three species is shown in Fig. 4. This figure indicates that the rat E90K:rat WT pair does not show dominant negativity, although both mouse E90K and human E90K GnRHR mutants show DN activity when co-expressed with the rat WT. This observation suggests that these human and mouse mutants are still able to interact with the rat WT, suggesting that the difference in the rat (compared with human or mouse) is not due to loss of the ability to oligomerize. Of interest, the human mutant is more effective as a DN regulator of the human WT and the mouse mutant is more effective in actions on both rodent WT receptors. Alteration of Amino Acid 216 in the Mouse GnRHR Mutant Creates a “Rat-like” Interspecific Structure and Increases Plasma Membrane Expression—Next, each of the four semi- or non-conservative amino acid changes (between the rodent species) in the mouse GnRHR(E90K) sequence were modified (singly) to make it more rat-like (Fig. 5A). Individual modification of three of these residues was uneventful; however, the substitution mGnRHR(E90K/G216S) resulted in markedly increased plasma membrane expression of the mouse (double) mutant. This interspecific construct had about 75% of the activity of the rat GnRHR(E90K). Consistent with expectations, modification of the rat GnRHR(E90K) sequence to make it more “mouselike” (S216G) is associated with a 2-fold loss of plasma membrane expression (Fig. 5B). Progressive modification of the remaining three semi- or non-conservative sites in the mouse GnRHR(E90K) sequence (with either G216S or Gly216) resulted in an unexpected loss of plasma membrane expression, suggesting a role for the conservative substitutions in the sequence (Fig. 5B). Because there are nine of these (Fig. 1, small boxes), further examination was not attempted in light of the earlier positive result. The Mutants in the Present Work Are Misrouted Proteins but Otherwise Fully Functional—It is conceivable that the effects observed with the derivative mutants of GnRHR(E90K) might reflect altered levels of mRNA expression, alterations in the ligand binding site or the sites that interact with effectors. To address these concerns, it was demonstrated that these mutants could be rescued with the pharmacoperone (IN3) to near wild-type levels (Fig. 6). Accordingly, these mutants are actually misrouted but rescuable and fully functional once restored to the plasma membrane. The DN Effect Is Also Lost Whenever the G216S Substitution Is Present—Fig. 7 shows the co-transfection of rat, mouse, and human WT GnRH receptors with mouse GnRHR(E90K) mutants in which the four rat-specific amino acids were altered singly or in combination. DN action was assessed in COS-7 cells. When mouse GnRHR(E90K) sequences contain the G216S mutation, the result is a loss in the ability of the mutant" @default.
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• W2016088181 title "Parallel Regulation of Membrane Trafficking and Dominant-negative Effects by Misrouted Gonadotropin-releasing Hormone Receptor Mutants" @default.
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• W2016088181 doi "https://doi.org/10.1074/jbc.m501978200" @default.
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