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Regulation of G Protein-coupled Receptor Trafficking by Inefficient Plasma Membrane Expression

2006; Elsevier BV; Volume: 281; Issue: 13 Linguagem: Inglês

10.1074/jbc.m510601200

1083-351X

Jo Ann Janovick, Paul E. Knollman, Shaun P. Brothers, Rodrigo Ayala-Yáñez, Abeer S. Aziz, P. Michael Conn,

Neuropeptides and Animal Physiology

Despite the prevalence of G protein-coupled receptors as transducers of signals from hormones, neurotransmitters, odorants, and light, little is known about mechanisms that regulate their plasma membrane expression (PME), although misfolded receptors are recognized and retained by a cellular quality control system (QCS). Convergent evolution of the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) progressively decreases inositol phosphate production in response to agonist, validated as a measure of PME of receptor. A pharmacological chaperone that optimizes folding also increases PME of human, but not of rat or mouse, GnRHR because a higher percentage of human GnRHRs are misfolded structures due to their failure to form an apparent sulfhydryl bridge, and they are retained by the QCS. Bridge formation is increased by deleting (primate-specific) Lys191. In rat or mouse GnRHR that lacks Lys191, the bridge is non-essential and receptor is efficiently routed to the plasma membrane. Addition of Lys191 alone to the rat sequence did not diminish PME, indicating that other changes are required for its effects. A strategy, based on identification of amino acids that both 1) co-evolved with the Lys191 and 2) were thermodynamically unfavorable substitutions, identified motifs in multiple domains of the human receptor that control the destabilizing influence of Lys191 on a particular Cys bridge, resulting in diminished PME. The data show a novel and underappreciated means of posttranslational control of a G protein-coupled receptor by altering its interaction with the QCS and provide a biochemical explanation of the basis of disease-causing mutations of this receptor. Despite the prevalence of G protein-coupled receptors as transducers of signals from hormones, neurotransmitters, odorants, and light, little is known about mechanisms that regulate their plasma membrane expression (PME), although misfolded receptors are recognized and retained by a cellular quality control system (QCS). Convergent evolution of the gonadotropin-releasing hormone (GnRH) receptor (GnRHR) progressively decreases inositol phosphate production in response to agonist, validated as a measure of PME of receptor. A pharmacological chaperone that optimizes folding also increases PME of human, but not of rat or mouse, GnRHR because a higher percentage of human GnRHRs are misfolded structures due to their failure to form an apparent sulfhydryl bridge, and they are retained by the QCS. Bridge formation is increased by deleting (primate-specific) Lys191. In rat or mouse GnRHR that lacks Lys191, the bridge is non-essential and receptor is efficiently routed to the plasma membrane. Addition of Lys191 alone to the rat sequence did not diminish PME, indicating that other changes are required for its effects. A strategy, based on identification of amino acids that both 1) co-evolved with the Lys191 and 2) were thermodynamically unfavorable substitutions, identified motifs in multiple domains of the human receptor that control the destabilizing influence of Lys191 on a particular Cys bridge, resulting in diminished PME. The data show a novel and underappreciated means of posttranslational control of a G protein-coupled receptor by altering its interaction with the QCS and provide a biochemical explanation of the basis of disease-causing mutations of this receptor. Pharmacological chaperones rescue misfolded mutants of the gonadotropin-releasing hormone (GnRH) 2The abbreviations used are: GnRH, gonadotropin-releasing hormone; GnRHR, GnRH receptor; PME, plasma membrane expression; QCS, quality control system; WT, wild type; ECL, extracellular loop; DMEM, Dulbecco's modified Eagle's medium; IP, inositol phosphate. receptor (GnRHR) isolated from patients with hypogonadotropic hypogonadism (1Conn P.M. Leaños-Miranda A. Janovick J.A. Mol. Interv. 2002; 2: 308-316Crossref PubMed Scopus (59) Google Scholar, 2Ulloa-Aguirre A. Janovick J.A. Brothers S.P. Conn P.M. Traffic. 2004; 5: 821-837Crossref PubMed Scopus (240) Google Scholar) and mutant proteins associated with other diseases (3Castro-Fernandez C. Maya-Nunez G. Conn P.M. Endocr. Rev. 2005; 26: 479-503Crossref PubMed Scopus (69) Google Scholar). For the GnRHR, such “pharmacoperones” are cell-permeant antagonists that promote correct folding of mutants, thereby allowing them to escape retention and degradation by the quality control system (QCS). Accordingly, these drugs are valuable for assessing whether loss of plasma membrane expression (PME) of particular mutants is due to misfolding in contrast to diminished mRNA expression, loss of ligand binding, or effector coupling. An unexpected finding was that pharmacoperones elevate plasma membrane expression of human, but not rat or mouse, WT GnRHRs (4Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar). This led to the consideration that the folding and structural requirements for hGnRHR are significant, more significant than those for the rodent GnRHR, consistent with a more rigorous control of ovulation in humans compared with rodents. In contrast, the rodent counterpart was more frequently correctly folded, passed quality control, and was trafficked to the plasma membrane. We confirmed this by identifying the biochemical structures that promote the destabilizing effect of primate-specific Lys191, which exerts its action by formation of misfolded receptors, thereby decreasing PME of this G protein-coupled receptor. The strong and convergent evolutionary pressure for what initially appears to be an increasingly inefficient process suggests a regulatory mechanism with considerable selective advantage. Materials—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 & Co.) (5Janovick 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, 6Ashton W. Sisco R. Yang Y. Lo J. Yudkovitz J. Gibbons P. Mount G. Ren R. Butler B. Cheng K. Goulet M. Bioorg. Med. Chem. Lett. 2001; 11: 1727-1731Crossref PubMed Scopus (40) Google Scholar), myo-[2-3H(N)]-inositol (NET-114A; PerkinElmer Life Sciences), DMEM, OPTI-MEM, Lipofectamine, phosphate-buffered saline (Invitrogen), competent cells (Promega, Madison, WI), and Endofree maxi-prep kits (Qiagen, Valencia, CA) were obtained as indicated. Mutant Receptor—Rodent and human WT and mutant GnRHR cDNAs for transfection were prepared as reported (4Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar); the purity and identity of plasmid DNAs were verified by dye terminator cycle sequencing (PerkinElmer). Transient Transfection—Cells were cultured in growth medium (DMEM, 10% fetal calf serum, 20 μg/ml of gentamicin) at 37 °C in a 5% CO2 humidified atmosphere. 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. 24 h after plating, the cells were washed once with 0.5 ml of OPTI-MEM and then transfected with 5 ng/well of WT or mutant receptor with 95 ng of pcDNA3.1 (empty vector) to keep the total amount of DNA at 100 ng/well. Lipofectamine was used according to the manufacturer's instructions. 5 h after transfection, 0.125 ml of DMEM with 20% fetal calf serum and 20 μg/ml of gentamicin were added. 23 h after transfection the medium was replaced with 0.25 ml of fresh growth medium. Where indicated, 1 μg/ml of IN3 in 1% dimethylsulfoxide (“vehicle”) was added for 4 h in respective media to the cells and then removed 18 h before agonist treatment (7Leaños-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). Inositol Phosphate (IP) Assays—27 h after transfection, cells were washed twice with 0.50 ml of DMEM/0.1% bovine serum albumin/20 μg/ml of gentamicin and “pre-loaded” for 18 h with 0.25 ml of 4 μCi/ml myo-[2-3H(N)]-inositol in inositol-free DMEM. The cells were washed twice with 0.30 ml of DMEM (inositol free) containing 5 mm LiCl and treated for 2 h with 0.25 ml of a saturating concentration of Buserelin (10–7m) in the same medium. Total IP was determined (8Huckle W. Conn P.M. Methods Enzymol. 1987; 141: 149-155Crossref PubMed Scopus (55) Google Scholar). This assay has been validated as a sensitive measure of PME for functional receptors when expressed at low amounts of DNA and stimulated by excess agonist (1Conn P.M. Leaños-Miranda A. Janovick J.A. Mol. Interv. 2002; 2: 308-316Crossref PubMed Scopus (59) Google Scholar, 2Ulloa-Aguirre A. Janovick J.A. Brothers S.P. Conn P.M. Traffic. 2004; 5: 821-837Crossref PubMed Scopus (240) Google Scholar, 3Castro-Fernandez C. Maya-Nunez G. Conn P.M. Endocr. Rev. 2005; 26: 479-503Crossref PubMed Scopus (69) Google Scholar, 4Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar, 5Janovick 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, 7Leaños-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, 9Cook J.V. Eidne K.A. Endocrinology. 1997; 138: 2800-2806Crossref PubMed Scopus (95) Google Scholar, 10Knollman P.E. Janovick J.A. Brothers S.P. Conn P.M. J. Biol. Chem. 2005; 280: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Janovick J.A. Ulloa-Aguirre A. Conn P.M. Endocrine. 2003; 22: 317-327Crossref PubMed Scopus (44) Google Scholar, 12Leaños-Miranda A. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 4825-4828Crossref PubMed Scopus (109) Google Scholar, 13Ulloa-Aguirre A. Janovick J.A. Leaños-Miranda A. Conn P.M. Expert Opin. Ther. Targets. 2003; 7: 175-185Crossref PubMed Scopus (31) Google Scholar). Binding Assays—Cells were cultured and plated in growth medium as described except that 105 cells in 0.5 ml of growth medium were added to 24-well Costar cell culture plates (cell transfection and medium volumes were doubled accordingly). 23 h after transfection, the medium was removed and replaced with 0.5 ml of fresh growth medium. 27 h after transfection, cells were washed twice with 0.5 ml of DMEM containing 0.1% bovine serum albumin and 20 μg/ml of gentamicin, and 0.5 ml of DMEM was added. After 18 h, cells were washed twice with 0.5 ml of DMEM/0.1% bovine serum albumin/10 mm HEPES, and a range of concentrations of 125I-Buserelin (1.25 × 105 to 4 × 106 CPM/ml) (4Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar) in 0.5 ml of the same medium were added to the cells. Cells incubated at room temperature for 90 min (14Brothers S.P. Janovick J.A. Maya-Nunez G. Cornea A. Han X.B. Conn P.M. Mol. Cell. Endocrinol. 2002; 190: 19-27Crossref PubMed Scopus (27) Google Scholar). After 90 min, the media were removed and radioactivity was measured as previously described (15Brothers S.P. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2003; 88: 6107-6112Crossref PubMed Scopus (49) Google Scholar). To determine nonspecific binding, the same concentrations of radioligand were added to similarly transfected cells in the presence of 10 μm unlabeled GnRH. Saturation binding curve fits and calculations were computed using SigmaPlot 8.02 (Jandel Scientific Software, Chicago, IL); a non-linear one-site binding model was used to calculate Bmax values (16Klotz I.M. Science. 1982; 217: 1247-1249Crossref PubMed Scopus (522) Google Scholar), which are displayed as percentages of respective WT levels. Statistics—Data (n ≥3) were analyzed with one-way analysis of variance and then paired Student's t test (SigmaStat 3.1; Jandel Scientific Software); p 88% Homologous yet Have Unique Features—Fig. 1 shows the hGnRHR, indicating residues that differ in the rat (ovals; the rat substitution is shown first in each pair of letters). Lys191 (absent in rat and mouse GnRHR) is associated with diminished PME of the human sequence and is shown by an enlarged circle in ECL2. The thermodynamic “favorability” of the interspecific substitutions (www.russell.embl-heidelberg.de/aas/aas.html, for membrane proteins) is indicated by numbers. Changes marked with zero are neutral substitutions, and positive numbers are favored. The three negative numbers (disfavored substitutions) are shown in bold and are summarized for other species in the table. Proximity suggests (9Cook J.V. Eidne K.A. Endocrinology. 1997; 138: 2800-2806Crossref PubMed Scopus (95) Google Scholar) that two Cys bridges may form in the rat and hGnRHR, connecting the NH2-terminal and ECL2 (Cys14-Cys199/200) and the ECL1 and 2 (Cys114-Cys195/196) as indicated. The Cys114-Cys195/196, but Not Cys14-Cys199/200, Bridge Is Required for Optimal Routing of the Rat and Mouse GnRHR, whereas Both Are Needed for hGnRHR—We assessed the potential bridge requirements by converting each of the four Cys to Ala (Fig. 2) in the mouse, rat, or human sequence. For all sequences, the bridge between ECL1 and ECL2 (Cys114-Cys195/196) appears essential because replacement of either Cys residue by Ala resulted in loss of IP in response to 10–7 m Buserelin (a saturating concentration of a metabolically stable agonist of the GnRHR) (10Knollman P.E. Janovick J.A. Brothers S.P. Conn P.M. J. Biol. Chem. 2005; 280: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). Under the conditions described under “Experimental Procedures,” the IP response of a functional receptor to a saturating concentration of agonist is a sensitive measure of PME (1Conn P.M. Leaños-Miranda A. Janovick J.A. Mol. Interv. 2002; 2: 308-316Crossref PubMed Scopus (59) Google Scholar, 2Ulloa-Aguirre A. Janovick J.A. Brothers S.P. Conn P.M. Traffic. 2004; 5: 821-837Crossref PubMed Scopus (240) Google Scholar, 3Castro-Fernandez C. Maya-Nunez G. Conn P.M. Endocr. Rev. 2005; 26: 479-503Crossref PubMed Scopus (69) Google Scholar, 4Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar, 5Janovick 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, 7Leaños-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, 9Cook J.V. Eidne K.A. Endocrinology. 1997; 138: 2800-2806Crossref PubMed Scopus (95) Google Scholar, 10Knollman P.E. Janovick J.A. Brothers S.P. Conn P.M. J. Biol. Chem. 2005; 280: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Janovick J.A. Ulloa-Aguirre A. Conn P.M. Endocrine. 2003; 22: 317-327Crossref PubMed Scopus (44) Google Scholar, 12Leaños-Miranda A. Janovick J.A. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 4825-4828Crossref PubMed Scopus (109) Google Scholar, 13Ulloa-Aguirre A. Janovick J.A. Leaños-Miranda A. Conn P.M. Expert Opin. Ther. Targets. 2003; 7: 175-185Crossref PubMed Scopus (31) Google Scholar). Pharmacoperone IN3 did not effect rescue (shown only for human, Fig. 2, inset). Replacement of either Cys in the bridge between the NH2-terminal and the ECL2 resulted in loss of IP production in the human but had a more modest effect in the mouse sequence and virtually no effect in the rat sequence. The human mutants (C14A or C200A) were rescued by IN3, suggesting that the absence of this bridge resulted in a rescuable misfolded protein. The Requirement for the Potential Cys14-Cys200 Bridge Is Lost in the Absence of Lys191 and Requires an Additional Motif Not Found in Rat or Mouse GnRHR—Deletion of Lys191 from the human WT sequence elevated PME, but insertion of Lys191 had little effect on the rodent receptors (which lack Lys191), suggesting that a more complex motif was responsible for the decreased efficiency of PME (Fig. 3A). Pharmacoperone IN3 increased PME of the hGnRHR (Fig. 2, inset), revealing that WT PME is limited by the presence of a proportion of misfolded receptors. Fig. 3B shows that removal of Lys191 from the hGnRHR sequence also rescues mutants (C14A and C200A), indicating a relation between these two sites and that the presence of Lys191 diminishes the probability of this association. Interestingly, the effect of the Lys191 is not wholly an effect of charge because converting it to uncharged Ala191, negatively charged Glu191, or modestly positively charged Gln191 also produces inefficient PME of human sequence compared with hGnRHR lacking Lys191, although to somewhat different levels (Fig. 3C). Particular Amino Acids Co-evolved with the Appearance of Lys191 or Other Insertions at Position 191—The table in Fig. 1 compares the most strongly, thermodynamically disfavored amino acid substitutions among GnRHR sequences for various species (amino acids 7, 168, and 203 (202 in rat and mouse)). Amino acid 189 was included because of its physical proximity to Lys191. Of the 40 amino acid differences between rodent and human GnRHR, it was striking that three of the four sites identified in the Fig. 1 table (amino acids 7, 168, and 189) also co-evolved with the insertion of an amino acid at position 191. The fourth (amino acid 202 in the rodent/203 in other species) changed in the human sequence. Exchange of Human and Rat ECL2 Appears to Influence the Proximity between Cys14 and Cys200—Fig. 4 shows the effect of mutating amino acids in the human second extracellular loop (hECL2) to the rat sequence (rendering the human ECL2 sequence more rat-like from amino acids 186 to 203), as well as several individual or multiple changes selected based on proximity to this region (Fig. 4A). The difference between the rat (Ala) and human (Thr) at position 190 was ignored because of the close conservation of these amino acids and the observation that mouse, guinea pig, canines, and ungulates are identical to humans at this site. The data show that replacement of the rat sequence by hECL2 enhances PME whether or not Lys191 is present (Fig. 4B, left). The reverse combination in which hECL2 is replaced by the rat ECL2 significantly blunts the effect of removal of Lys191 and diminishes the overall level of PME (Fig. 4B, right). These results were unexpected because the rat WT normally expresses at a higher level than the human WT. Such mutants (including the human GnRHR with mutation to the rat sequence at ECL2 alone (Fig. 4B), 168/ECL2, 161/168/ECL2, 182/ECL2, 189, 186, 203, or 189/203, Fig. 4D) are rescued with pharmacoperone IN3 (not shown) to levels above that observed for WT receptor. Moreover, deletion of Lys191 (Fig. 4, B and D), a modification associated with increased routing to the plasma membrane, also rescues these mutants. These observations confirm that the effects of mutating ECL2 are due to misfolding and misrouting rather than altered interactions with effectors or altered levels of mRNA synthesis. Also surprising, replacement of the rodent ECL2 with the human sequence did not restore the ability of Lys191 to interfere with folding or routing and actually increased the level of PME. Mutations in Amino Acids within ECL2 or in Structures Flanking It Also Regulate the Relation between the NH2-terminal and ECL2—The effects of mutating individual amino acids within and flanking ECL2 were examined (Fig. 4C, rat; Fig. 4D, human) because these might impact the relation between Cys14 and Cys199/200 and regulate the probability of bridge formation. Making the hGnRHR rat-like at position 168, 182, 189, or 203 did not have a marked effect on the (otherwise) WT receptor. Changes at 161 or 186 increased PME of the WT sequence. When Lys191 was deleted from the hGnRHR sequence a second mutation at position 161, 168, 182, 186 or 203 exacerbated the effect of the deletion, but mutation at 189 alone or the combination of ECL2 with 168 diminished the increased PME observed due to deletion of Lys191 to near WT levels. Likewise, the combined mutation of 189 and 189/203 resembled the effect of mutating ECL2 only. Finally, the human sequence (Fig. 4D) tolerated changes from amino acids 155–161, further from Cys200, including the effect of Lys191 removal, unless these were combined with the rat ECL2. Combining changes at positions 161 and 168 (in effect, moving the rotational influence of Ser on the protein backbone further from Cys200) with ECL2 resulted in loss of activity of WT and the Lys-deleted sequence. In the rat, mutations at 161,168, 182, 186, and 189 increased WT PME (Fig. 4C). Less dramatic increases were seen with mutation in position 202 of rat GnRHR. When combined mutants (189/202, 168/ECL2, or 182/ECL2) were made, the effect of the mutant that exacerbated PME appeared to predominate. Also in the rat sequence, several mutants into which Lys191 was inserted showed higher PME (161, 168, 189, 202); 182 and 186 showed more modest effects. The proximity required for Cys bonds to form (1–2 Å) explains why exceedingly precise alignment is needed for Cys14-Cys200 and why twisting of the support sequences (transmembrane segments 4, 5) or those that abut on that area (i.e. ECL1, 3) also impacts bridge formation. Other Sites Impact on the Net Level of Receptor PME and Allow the Effect of Lys191—Modifications of the hGnRHR at positions 4, 7, and 10 resulted in PME above the level of WT and amplification of the effect of deletion of Lys191 (Fig. 5A). Mutation at these same sites in the rat GnRHR did not have a measurable effect on WT PME, although modifications at 4, 7, or 10 in the presence of hECL2 enhanced PME. Mutations in human positions 24 and 27 (Fig. 5A) decreased PME of WT GnRHR and blunted the effect of deletion of Lys191. In the rat, homologous mutations at residues 24 and 27 increased WT and WT(+Lys191) PME, perhaps due to the replacement of a Thr (Cβ-branched, presenting bulkiness near the backbone) with an amphipathic Met24 that allows the side chain to be buried and increases the chance of bridging the NH2-terminal with ECL2. We created an additional series of interspecific chimeras. Fig. 5, B (rat) and C (human), shows the dramatic effect of modifications at amino acids 112, 207/208, 224/225, 299/300, 301/302, as well as selected combinations of these sites. Unlike the majority of mutations associated with ECL2, these are all steric in nature, because hydrophobicity and charge are largely conserved. In the rat sequence, mutations were made at 299 alone or in combinations. For all mutants PME was blunted. Insertion of Lys191 with 299 mutants also had diminished PME. Mutations at 207, 224, 301 were also associated with elevated PME, with or without Lys191. Of note, substitutions at positions 161, 207 224, 207/224, and 229/301 (Figs. 4 and 5) each allowed destabilization by Lys191 in the rat sequence, presenting the possibility that contributions from multiple sites were important. Individual homologous mutations in the human sequence did not totally ablate the ability of the deletion of Lys191 to decrease PME; some actually increased the effect, again consistent with the view that several different sites contribute to the final effect of Lys191. In addition, mutations at 300 and 302 dramatically increased overall PME in the human sequence, with or without Lys191, and decreased the same in the rat sequence. Breaking the Cys14-Cys200 Bridge Allows Assessment of the Stability of the Association of the NH2-terminal and ECL2 in Human and Rat Mutants—To determine the stability of the NH2-terminal-ECL2 relation and the persistence of destabilization by Lys191 created by the transfer of human substitutions to the rat sequence, we prepared “broken bridge” mutants (i.e. C14A, Fig. 5D) for rat and human sequences indicated by brackets (Fig. 4, B–D, and Fig. 5, B and C). The human sequence in which position 189 was converted to rat (Q189P) results in modestly more PME than hWT and a considerable blunting of the effect of removal of Lys191 (Fig. 4D). When the bridge is broken in this sequence, the ability to create a functional receptor is lost and not rescuable by deletion of Lys191. This observation means that formation of the bridge occurs at a time when the association of these regions is intimate. The observation that all rat sequences in which we successfully transferred the destabilizing effect of Lys191 with human substitutions lose this effect when the bridge is unable to form (Fig. 5D, C14A) suggests that failure to create the bridge during synthesis results in a lost opportunity for Lys191 to modulate this effect. The impact of Lys191 can only occur at the time when the bridge is able to form (likely at the time of synthesis) and not once the molecule is complete. The human with rat substitutions at positions 112, 208, 300, and 302 is remarkable, as these amino acids flank and appear to stabilize the association of positions 14 and 200 even in the absence of the Cys bridge (i.e. when C14A is present). In this curious molecule, which is primarily human, we observe rat-like PME levels (rat WT GnRHR typically reaches the plasma membrane with about twice the efficiency of the human WT) and lack of requirement for the Cys14-Cys200 bridge yet maintenance of sensitivity to the effect of destabilization by Lys191. Quantification of Plasma Membrane GnRHRs by Radioligand Binding—To confirm that the results measured in the functional assay (IP production) reflected changes in actual receptor numbers (in contrast to changes in the efficiency of coupling to G proteins, for example), we used radioligand binding to quantify receptor numbers of selected WTs and mutants (Fig. 6). We included rat and human WT GnRHR, as well as human WT (which normally contains Lys191) with this residue deleted, and rat WT (which normally does not contain Lys191) with this residue added. We also included representative protein-encoding plasmids with the exchange of the human and rat ECL2 for both species, as well as representatives of human sequences with C14A. This modification results in breaking the apparent bridge between positions 14 and 200, a modification that is without effect in the rat. We also included human mutants in which the rat characteristics (less requirement for the Cys bridge and higher expression levels) were conveyed (112/208/300/302). The data confirm radioligand binding and IP production produce similar conclusions, notably that deletion of Lys191 from the hWT markedly increases PME, but adding this residue to the rat has little effect. Further, breaking the apparent Cys14-Cys200 bridge with the mutation C14A is associated with loss of PME in the human unless the 112/208/300/302 mutation is present. Lys191 in the hGnRHR sequence (328 amino acids) appears to raise the folding criteria and structural requirements for the hGnRHR compared with its rat/mouse counterparts that lack this residue (327 amino acids) or hGnRHR from which Lys191 was deleted (1Conn P.M. Leaños-Miranda A. Janovick J.A. Mol. Interv. 2002; 2: 308-316Crossref PubMed Scopus (59) Google Scholar, 2Ulloa-Aguirre A. Janovick J.A. Brothers S.P. Conn P.M. Traffic. 2004; 5: 821-837Crossref PubMed Scopus (240) Google Scholar, 3Castro-Fernandez C. Maya-Nunez G. Conn P.M. Endocr. Rev. 2005; 26: 479-503Crossref PubMed Scopus (69) Google Scholar, 4Janovick J.A. Maya-Nunez G. Conn P.M. J. Clin. Endocrinol. Metab. 2002; 87: 3255-3262Crossref PubMed Scopus (169) Google Scholar). The view that Lys191 increases the percentage of misfolded/misrouted receptors is supported by the observation that a pharmacoperone, which corrects folding errors, enabling mutants to pass through the cellular QCS, also increases the PME of the human WT, but not rat GnRHR. This observation also allowed us to exclude ligand binding, effector coupling, or level of mRNA expression as a cause of reduced mutant PME (10Knollman P.E. Janovick J.A. Brothers S.P. Conn P.M. J. Biol. Chem. 2005; 280: 24506-24514Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 11Janovick J.A. Ulloa-Aguirre A. Conn P.M. Endocrine. 2003; 22: 317-327Crossref PubMed Scopus (44) Google Scholar). Radioligand binding data and studies in which pharmacoperones are used to correct folding and sequential routing indicate that, at the low cDNA levels transfected, IP production is a good measure of functional receptors at the plasma membrane. Removal of Lys191 or use of pharmacoperone obviates the requirement for an apparent Cys14-Cys200 bridge in the hGnRHR required for correct folding. Because inserting Lys191 alone into the rat or mouse sequence (>88% homologous with the human) did not impart the requirement for this bridge between the NH2-terminal and ECL2, we sought to identify the other components of the requisite motif. We examined thermodynamically unfavorable amino acid substitutions after recognizing that these frequently co-evolved with the appearance of the “extra” amino acid at position 191 and were proximal to it and to the bridge. An extra amino acid at position 191, not necessarily Lys, is present in all non-rat/mouse mammalian species that have been sequenced to date. Insertion of Lys191 or Glu191 is associated with substitution of Leu7 for Pro7, except in which case a less favorable change is in the guinea pig made (Ser7). Furthermore, Lys191 is associated with a change from Ile168 to Ser168 in primates, whereas Glu191 is associated with Ser168 in the guinea pig an