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J. Biol. Chem., Vol. 281, Issue 13, 8417-8425, March 31, 2006
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¶1
From the
Divisions of Neuroscience and Reproductive Biology, Oregon National Primate Research Center, and Departments of Physiology and Pharmacology and ¶Cell and Developmental Biology, Oregon Health and Science University, Beaverton, Oregon 97006
Received for publication, September 28, 2005 , and in revised form, January 27, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Mutant Receptor—Rodent and human WT and mutant GnRHR cDNAs for transfection were prepared as reported (4); 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 x 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 (7).
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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 x 105 to 4 x 106 CPM/ml) (4) in 0.5 ml of the same medium were added to the cells. Cells incubated at room temperature for 90 min (14). After 90 min, the media were removed and radioactivity was measured as previously described (15). 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 (16), 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 <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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.
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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 (C
-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.
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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.
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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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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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 and ungulates but Gly168 in the case of canines. Such substitutions are always unfavored. Ile is C branched with two non-H substituents attached to the carbon, presenting bulkiness near the protein backbone and restricting conformations that the main chain can adopt.
Insertions at position 191 also correlate with a change of the Pro189 to Gln189, neutral for membrane proteins but unfavored (–1) in the extracellular space. Only primates show the change from Pro202/203 to Ser203.
Glu 191is found in the sequence of many pre-primate mammalian GnRHs (Fig. 1, table). Although it too can destabilize bridge formation, it is markedly less effective than Lys191 found in primates. This further supports the progressive trend of limiting GnRHR expression at the plasma membrane in higher evolved species.
The effect of mutations associated with human disease emphasizes the importance of the Cys14-Cys200 bridge. Mutants S168R and S217R are in a previously reported "zone of death" (1) and cannot be rescued by any of several different chemical classes (indoles, quinolines, and erythromycin macrolides) of pharmacoperones tried, which is a rare circumstance because the vast majority of mutants are rescuable by all classes (5). This observation and the physical relation between transmembrane segments 4 and 5 and ECL2 make it attractive to consider that (charge-altering) mutations in these two residues exert their influence by regulating the position of ECL2 and the intimacy of Cys14 and Cys200. Because of charge considerations, the unfavored exchange of Ser and Arg likely moves the ECL2 into a position from which the formation of a Cys bridge is improbable and the mutant never passes the cellular QCS even in the presence of pharmacoperones. The dramatic effect of the mutation at position 217 supports the view that the Cys14-Cys200 bridge is not created until the transmembrane segment 5 is formed.
The importance of position 217 is emphasized by recognizing that the homologous residue in rodents (amino acid 216) is associated with substantial differences in both trafficking and dominant negative action between the rat (Ser216) and mouse (Gly216) GnRHR (10), despite >96% homology. The substitution Ser217 with a highly flexible Gly217 results in loss of PME of the mutant, an effect that is rescuable by deletion of Lys191 (10).
C200Y is another disease-associated mutation (12–13, 17–19) that results in loss of the Cys14-Cys200 bridge and decreased PME; rescue by a pharmacoperone (10–13) supports the significance of the bridge for creating a correctly folded moiety. Consistent with that view, the homologous mutation in the rat or mouse GnRHR is a fully functional receptor. Indeed, the observation that many human disease-associated mutations recreated in rodent templates are functional (10) emphasizes the increased significance of the QCS in the human. It appears that a balance is created (in the human) between retention by the QCS and routing to the membrane. Accordingly, the human, but not rat or mouse, GnRHR is extremely sensitive to naturally occurring point mutations (10) that perturb this balance.
Of rodents, animals with large litters, only the guinea pig is known to have added an amino acid (Glu) at position 191 of the GnRHR. Interestingly, the guinea pig, a hystricomorph that diverged very early in rodent evolution, has a long luteal phase (a primate characteristic). Most non-rodent mammals such as cows, sheep, pigs, dogs, and horses also contain Glu191, suggesting that the loss of an amino acid in the homologous position is a specialization associated with very short reproductive cycles in rats and mice. Unlike all other reported mammalian sequences, the opossum (a non-placental mammal that places fetuses in a marsupium) has an uncharged, racemic Gly191 that may reflect the early divergence of this group. In hGnRHR, substitution of uncharged (Ala191) or negatively charged (Glu191) amino acids appears to have similar effects as the Lys191, both in the epitope-tagged (20) and in the WT hGnRHR (present study), a finding that suggests that neither charge nor specific steric considerations totally explain the action of this amino acid but supports the view that the extra amino acid provokes crowding and a change in the relative positioning of amino acids that normally leads to Cys bridge formation. Noting that various species have inserted Lys191, Gly191, or Glu191 suggests convergent evolutionary pressure for a means to use this position as a regulatory mechanism.
The observation that residues that regulate the efficiency of trafficking continue to evolve, even among the upper primates, suggests that selective pressure remains for this regulatory approach. Ser203 and Leu300 are uniquely human as other mammals have Pro203 or Val300. Asp302 is found in mammals with an extra amino acid at position 191. In rodents the corresponding amino acid is Glu301, believed to interact with the Arg8 of the natural ligand, GnRH (21). Position 112 is Phe in rat, mouse, opossum, dog, and pig, but the human sequence contains Leu112 at this position. This is not a case of uniqueness, because cows, sheep, horses, and even pre-mammals (fruit fly, chicken, and Xenopus) share Leu at this site.
In considering the molecule as a whole, the modifications associated with orientation of ECL2 (positions 7, 168, 189, and 202/203) all involve the gain or loss of Pro or Ser. Pro forms a five-membered nitrogen-containing ring, a feature that causes it to be found in very tight turns in protein structures (i.e. where the polypeptide chain must change direction). Ser, with a slightly polar nature, small size, and propensity of the side-chain hydroxyl oxygen to H-bond with the protein backbone, also causes it to be found in association with tight turns of the protein structure.
Several conservative modifications were present in regions that might impact the putative Cys14-Cys199/200 bridge. These modifications appear to participate in recreating the bridge-destabilizing effect of Lys191 in the rodent, suggesting that the effect in human is the impact of multiple Among the conserved modifications, evolutionary changes. Leu224/225 was changed to Phe225 only in canines, whereas only in humans was Val300 converted to Leu. There also appeared to be a relation between Asp301/302 because this residue is Glu301 in rodents lacking an insertion at position 191. The ability of particular residues to have an impact at such an apparent distance is both a tribute to the interactive nature of this G protein-coupled receptor and the failure of two-dimensional representations to portray accurately the proper spatial relations.
The progressive decrease of PME within mammals began in premammalian classes. The GnRHR in fish and birds (species that produce large numbers of offspring at low metabolic energy per unit and low survival and have a single gonadotropic hormone) contains a cytoplasmic carboxyl-terminal tail that increases net expression and effector coupling efficiency of the receptor at the plasma membrane (11, 22). Truncation of the tail in these species results in diminished PME, whereas production of a chimeric structure of a rodent GnRHR containing the catfish tail increases PME (11, 22). Piscine and avian GnRHRs also lack the cysteines needed to bridge the NH2-terminal region and the ECL2; such residues are absolutely conserved among mammalian GnRHRs. Because the number of receptors per cell expressed on the plasma membrane (23, 24) cycles and thereby controls gonadotropin levels and in turn ovulation, it may be that the advent of complex primate ovarian cycling requires the ability to regulate promptly the GnRHR without the requisite time for translation and transcription.
The observation that hGnRHR is inefficiently expressed at the plasma membrane is intriguing, because this is the result of a progressive and convergent evolutionary trend toward diminished PME of the GnRHR, adds metabolic cost to create unused receptors, and creates an increased sensitivity to mutation. Potentially, this mechanism provides a source of GnRHR needed for rapid availability without transcription or translation. A similar mechanism appears to regulate the human 
opioid receptor because permeable agonists and antagonists also facilitate post-translational processing and increased export of the ligand-stabilized receptor from the endoplasmic reticulum to the cell surface (25). Other reports indicate that other receptors (GluR1, 
1D adrenoreceptor, odorant, and Luteinizing hormone receptors) are likewise inefficiently expressed at the plasma membrane (26–30); this suggests that restricted trafficking may be a more commonly occurring means of regulating protein availability than presently appreciated.
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FOOTNOTES |
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1 To whom correspondence should be addressed: ONPRC/OHSU, 505 NW 185th Ave., Beaverton, OR 97006. Tel.: 503-690-5297; Fax: 503-690-5569; E-mail: connm{at}ohsu.edu.
2 The 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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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