Parallel Regulation of Membrane Trafficking and Dominant-negative Effects by Misrouted Gonadotropin-releasing Hormone Receptor Mutants*

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 Ser 216 in the rGnRHR. The homologous amino acid in the hGnRHR is also Ser and is compensated for by the primate-unique insertion of Lys 191 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 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 (30) 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 (cid:5) 0.05 considered significant. One-way analysis of variance was used to determine interexperimental variance between data sets.

Not surprisingly, pharmacoperones also reverse the DN effect (24,25) 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 Glu 90 3 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, Lys 191 , that is inserted in the human receptor sequence (328 amino acids) (20) was examined. The presence of Lys 191 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 Lys 191 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.
Transient Transfection and Co-transfection-Cells were cultured and plated in growth medium (DMEM, 10% fetal calf serum, 20 g/ml gentamicin), and growth conditions were 37°C and 5% CO 2 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 ϫ 10 4 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 (pcDNA3.1ϩ without insert "empty vector" was included to bring the total cDNA to 100 ng/well, unless otherwise indicated) and 1 l 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 g/ml gentamicin was added to the wells. Twentythree h after transfection, the medium was removed and replaced with 0.25 ml of fresh growth medium. Where indicated, 1 g/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 (3,5,24). Data are presented as the means Ϯ 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 g/ml gentamicin and then "preloaded" for 18 h with 0.25 ml of 4 Ci/ml myo- [2-3 H]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 Ϫ7 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 (30) 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 Ͻ 0.05 considered significant. One-way analysis of variance was used to determine interexperimental variance between data sets. 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, Lys 191 (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.

Structural Features of the GnRHR-
Substantial Evolutionary Homology between the Rodent and Human GnRHR- Fig. 1B (top right) also shows a phylogram tree constructed from the reported sequences (2) 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 -10 for vector-only control values). 10 Ϫ7 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 Ͻ 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.
Lys 191 , 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 (25). Fig. 3 also shows the effect of deleting the Lys 191 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 Lys 191 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 Lys 191 insertion. In both rodent sequences, expression of E90K mutants was reduced by the Lys 191 insertion. The rat GnRHR(E90K) mutant expresses 15.06 ϩ 0.32% compared with the WT, followed by very modest expression of the mouse GnRHR(E90K) mutant (3.59 ϩ 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 mem-brane expression (Fig. 5B). Progressive modification of the remaining three semi-or non-conservative sites in the mouse GnRHR(E90K) sequence (with either G216S or Gly 216 ) 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 FIG. 1. A, GnRHR map showing the four semi-or non-conservative locations in mouse and rat GnRHR, as well as the location of particular amino acids. Of the 13 amino acid residue differences between mouse and rat GnRHR sequences, there are nine conservative differences (small boxes with single letter shows the mouse substitution) and four semi-or non-conservative differences. The semi-or non-conservative locations (amino acids 11, 24, 160, and 216) are shown by shaded boxes that also compare the human, mouse, and rat substitutions at those positions. Evolutionary insertion of primate-specific Lys 191 (indicated with a circle and arrow) to the human GnRHR results in amino acid 217 corresponding to amino acid 216 in the mouse and rat GnRHR domain. The large box compares the human, mouse, and rat sequences in the fifth transmembrane sequence, showing position 216/217 in bold. The extracellular (EC) and intracellular (IC) plasma membrane orientation is shown. The position of amino acid Glu 90 in the second transmembrane portion of the molecule is indicated with a circle and arrow. B, the phylogram tree, showing the relation between the GnRHR from human, mouse, rat, and other species, was produced from reported protein sequences using Clustalw online at www.ebi.ac.uk/clustalw/. 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 mouse sequence to produce a significant DN effect on WT GnRHR. When other "rat" mutants are created that result in making the mouse GnRHR(E90K) sequence more rat-like, the DN effect is also lost whenever the G216S substitution is present.
Functional Relation between Two Amino Acids-The effect of inserting the Lys 191 residue in rat and mouse WT GnRHR sequences, as well as in the G216S (mouse) and S216G (rat) or S217G (human) mutants (Fig. 8A), was studied. Both mouse and rat WT GnRHR(ϩLys 191 ) mutants show no altered IP production as compared with respective WT. However, when the rat sequence contains the S216G mutation, the insertion of Lys 191 results in decreased IP production. Additionally, when the mouse G216S mutant contains the inserted Lys 191 , IP production is decreased. This suggests a possible unexplained relation between amino acids 191 and 216. As expected, both human WT GnRHR and human GnRHR(S217G) show increased IP production when the Lys 191 residue is removed (des-Lys 191 ). B-D show the result of co-transfection of the GnRH mutants in Fig. 8A with indicated WT (listed above graphs). No DN action was seen, but rather an additive effect was noticed in all cases. These observations suggest that these mutants do not oligomerize (as seen with the GnRHR(E90K) mutants) but rather express both WT and mutant GnRH at the plasma membrane.
Reconstruction of Human Disease-causing Mutants in Rodent Sequences Shows That the Rodent Sequences Are Less Easily Destabilized by These Mutants- Fig. 9 shows the effect of constructing a number of human mutations in the rodent GnRHR sequences (A and B). These point mutations were isolated from patients with hypogonadotropic hypogonadism. For comparison, the human data are shown (C). The pharma-  3. IP production in response to rat, mouse, and human WT GnRHR and GnRHR mutants containing ؎ Lys 191 and E90K, alone or in combination. 95 ng of the indicated sequence was expressed with 5 ng of vector (total 100 ng of DNA) in COS-7 cells, and IP production in response to 10 Ϫ7 M Buserelin was measured. When 100 ng of empty vector was transfected and treated similarly, the result was indistinguishable from that measured with hGnRHR(E90K). Columns marked with an "ϫ" were not measured, as they denote a GnRHR sequence identical to the individual species WT or E90K mutant sequences; Lys 191 is naturally occurring in primate GnRHR and was added in this study to rodent GnRHR sequences. The data are represented as the mean Ϯ S.E. of three independent experiments, each performed in replicates of six. *a, p Ͻ 0.05 compared with rGnRHR WT IP production. *aa, p Ͻ 0.05 compared with rGnRHR(E90K) IP production. *b, p Ͻ 0.05 compared with mGnRHR WT IP production. *bb, p Ͻ 0.05 compared with mGnRHR(E90K) IP production.*c, p Ͻ 0.05 compared with hGnRHR WT IP production. *cc, p Ͻ 0.05 compared with hGnRHR(E90K) IP production. coperone, IN3, is used to restore proper folding and thereby rescue the misrouted mutant receptor. In the case of the rat GnRHR, the ability of these mutations to cause misrouting of the rat mutant is lost or greatly diminished (compared with their effects on hGnRHR), except in the mutations S216R, L265R, and Y283C. The rodent GnRHRs appear to be more tolerant of mutation than is the human GnRHR. Additionally, the mouse seems less forgiving than the rat to the HH substitutions, as the mouse mutations more greatly reduce plasma membrane expression of the mutant GnRHRs (compared with the rat). This observation validates the physiological significance of the small number of semi-and non-conservative differences between the mouse and rat GnRHRs. Fig. 10A shows the effect of constructing the four semi-and non-conservative rodent differences in the mouse WT GnRHR, with the rat GnRHR(S216G) shown on the left for comparison. Unlike the effect seen in mutants, there were only very modest differences between any of the singly or multiply modified mouse mutants in comparison with mouse WT GnRHR plasma membrane expression levels. This suggests that the difference between the rat and mouse at position 216 has no effect on wild-type expression and is only observed when mutations are present that decrease expression at the plasma membrane. This is not surprising in light of the view that both mouse and rat GnRH wild-type receptors appear to express fully (or nearly so) the translated FIG. 4. Specificity of rat, mouse, and human E90K mutants to exert a DN effect on WT receptors from these species. WT receptor (5 ng) from each of the three species (listed above each graph in a box) was co-transfected with vector or E90K (95 ng, 100 ng of total DNA) homolog corresponding to the rat, mouse, or human GnRHR sequence. IP production in response to 10 Ϫ7 M Buserelin was assessed as described under "Experimental Procedures." The data are represented as the mean Ϯ S.E. of at least five independent experiments, each performed in replicates of six. *a, p Ͻ 0.05 compared with co-transfection of rWT GnRHR and vector. *b, p Ͻ 0.05 compared with co-transfection of mWT GnRHR and vector. *c, p Ͻ 0.05 compared with co-transfection of hWT GnRHR and vector.

Modification of Amino Acid 216 in the Wild-type Rodent Sequences Suggests That This Site Functions to Stabilize Partially Expressed Sequences but Has No Effect on Highly Expressed Rodent Wild-type GnRHR-
FIG. 5. Modification in the mouse GnRHR sequence at nonconservative sites, corresponding to the differences found in the rat sequence. A, the effect of individually mutating the four semior non-conservative site differences between mouse and rat WT receptors (amino acids 11, 24, 160, and 216) in the mouse GnRHR(E90K) sequence. For comparison the mouse and rat GnRHR(E90K) sequences (without further modification) are shown. B, once the importance of amino acid 216 was recognized, multiple non-conservative site mutants were created in the mouse GnRHR(E90K) sequence with the naturally occurring Gly 216 sequence (black data bar) and the mutant G216S sequence (white and black data bar). It should be noted that the rat GnRHR(E90K) contains the native Ser 216 and appears as the stacked black and white bar. The modification S216G in the rat GnRHR(E90K) sequence is shown as only the black bar on the same column. A/B, transfected COS-7 cells (95 ng of mutant or WT with 5 ng of vector) were stimulated with 10 Ϫ7 M Buserelin, and IP production was measured. Data represent the mean Ϯ S.E. of at least three independent experiments, each performed in replicates of six. *aa, p Ͻ 0.05 compared with rGnRHR(E90K). *bb, p Ͻ 0.05 compared with mGnRHR(E90K).
WT receptor at the plasma membrane. Consistent with these expression results, no DN effects were seen (B-D) with the mouse mutants on any of the three species WT GnRHR. DISCUSSION The present data indicate that a single amino acid, Ser 216 , in the rat GnRHR sequence increases the efficiency of routing to the plasma membrane of rat mutants that are otherwise misrouted. This same amino acid is responsible for loss of the DN effect of rat mutants when co-expressed with WT receptor. In the human sequence, also containing a Ser in the homologous position (Ser 217 ) this effect is mitigated by the presence of an "extra" amino acid, Lys 191 , inserted in the primate sequence and not present in earlier species. Concerns for effects of mutation on altered levels of mRNA expression, ligand binding, or effector coupling have been eliminated by demonstration that the mutants used in the present study are rescuable by a pharmacoperone and are restored to nearly identical activity levels compared with the wild-type receptor. The finding that these mutants become fully functional proteins indicates that loss of activity is due to misrouting. The observation that the rat WT GnRHR retains the ability to oligomerize with mutants derived from human and mouse GnRHRs suggests that the effect on the rat mutants is also likely due to altered trafficking rather than loss of the ability to oligomerize.
Nature has restored the DN effect in primates by inserting Lys 191 (along with Ser 217 ), which greatly diminishes expression of the human sequence at the plasma membrane (31), and greatly increases the susceptibility of disturbing the conformation of this receptor with single charge changes in the primary sequence.
In humans, the rat Ser 216 homologous locus is also the site of a naturally occurring mutation in the human receptor that leads to hypogonadotropic hypogonadism (S217R). This mutation is one of two known mutations that cannot be rescued by pharmacological chaperones (3), suggesting that either such mutations have a dramatic effect on the conformation of the GnRHR or that this area is important for ligand binding or effector activation. Most models exclude the second possibility (25,32). In addition, because the Ser 216/217 is within the membrane (fifth transmembrane domain), it is not likely that this is a phosphorylation site; moreover it is not within a known consensus phosphorylation sequence (box, Fig. 1). It is not surprising that even a residue that will eventually end up in the plasma membrane can interact with the quality control apparatus of the cell in light of the observation that many naturally occurring mutants have this action (5).
It is interesting to consider the biochemical nature of the mutations that can lead to the HH disorder. At the time this was being written, there are 18 mutants (5-6, 12-23) known to cause isolated HH. Of these, two are missing large sequences: one being a truncation of all amino acids between 314 and the carboxyl-terminal amino acid 328 (19) and the other a deletion mutant missing exon 2 (33,34). It is reasonable to assume that such large sequence omissions would have a dramatic effect on the receptor structure. The remaining mutations are subtler, involving only a single amino acid. Of these, three involve loss (two occurrences) or gain (one occurrence) of a cysteine residue, an amino acid known to form bridges associated with the formation of third order protein structure. Disruption of required bridges or formation of inappropriate bridges would be significantly disruptive to the protein structure. One of the recently reported mutations (35) is the loss of a proline at position 320, which is replaced by a leucine. Because the peptide backbone of proline is constrained, occurrence of this imino acid is associated with a forced turn in the protein sequence. Although this is a hydrophobic for hydrophobic substitution, the turn is a likely requisite for receptor conformation involved in routing or activity and cannot be corrected by the pharmacoperones. The remainder of the HH-associated mutants, fully 12 mutants, are caused by modest changes in a single charge. Ten of these 12 mutations involve lysine (three occurrences), arginine (six occurrences), or aspartic acid (one occurrence). Introduc-tion of charge changes (even minor) appear sufficient to alter the structure. Of interest, none of the reported mutations are conservative in which, for example, alanine replaces a glycine or threonine replaces a serine; in each case adding a single carbon without modifying the net charge. Likewise, there are no examples of simple hydrophobic for hydrophobic exchanges (valine for alanine, for example), positive for positive (lysine for arginine) exchanges, or negative for negative (aspartic acid for glutamic acid) exchanges. Such exchanges may occur, of course, but may be clinically silent, or alternatively, the genotype may not be propagated into subsequent generations.
In the case of the mutant positions in human receptor associated with disease, the amino acid in the corresponding human WT is significantly conserved in mammals. The exceptions are Asn 10 3 Lys (for which aspartic acid is found in mouse and rat but asparagine in other mammals sequenced), Ser 168 3 Arg (for which isoleucine is found in mouse and rat but serine in other mammals sequenced), Ser 217 3 Arg (mouse has glycine; guinea pig has isoleucine), Cys 200 3 Tyr (glycine in pig), Tyr 284 3 Cys (leucine in pig). The Arg 139 3 His is part of the DRS motif (DRY in most mammalian receptors) in intracellular loop 2.
Nature likely relies on this remarkable charge sensitivity of the human GnRHR to diminish the efficiency of transfer of this protein to the plasma membrane by insertion of Lys 191 . Pharmacoperone rescue studies suggest that insertion of this charged amino acid decreases the expression of the human WT receptor by about 40% (31). The ability of pharmacoperones to elevate the plasma membrane expression of WT human Gn-RHR (WT contains Lys 191 ), but not rodent GnRHR (WT does not contain Lys 191 ), and the location in the receptor (second extracellular loop) suggests that the occurrence of this added amino acid is associated with regulation of routing in contrast to alteration in the coupling mechanism, for example. For reasons that are buried in the physically diffuse amino acid differences between the rodent and human sequence, the simple addition of Lys 191 to WT rodent GnRHR does not result in the expected diminution of routing (31).
Even before mammals, there has been a progressive trend toward a decrease in the percentage of the synthesized GnRHR receptor that is expressed at the plasma membrane. In fish and avians, the GnRHR homolog contains a long carboxyl-terminal tail associated with increased expression (36). Human or rodent chimeras containing this "C-tail" sequence route more efficiently to the plasma membrane than do the WT molecule lacking it (37,38).
While the reason for this progressive loss of efficiency of trafficking to the plasma membrane is not clear, it may be associated with the added complexity and cyclicity of the reproductive process in mammals and, subsequently, in primates (39 -42). In rodents and primates, the concentration of the GnRHR on the plasma membrane cycles, and a store of synthesized receptor may be valuable to call upon promptly and without the delay required for further transcription or translation. The added reliance of the human receptor on endoplasmic reticulum-resident protein chaperones may provide an important site of regulation.
These studies establish the relationship between the DN effect and altered receptor trafficking and explain why the delicately balanced plasma membrane expression of the human GnRHR is more susceptible to defective trafficking as a result of point mutations than its rat and mouse counterparts. The important role of protein routing is well established in biological systems (27)(28)(29)43). The present work indicates a progressive evolution toward the regulation of trafficking of a particular protein to the plasma membrane and presents the WT GnRHR sequences were transfected in COS-7 cells (95 ng of WT with 5 ng of vector DNA) and stimulated with 10 Ϫ7 M Buserelin, and IP production was measured. Data represent the mean (n ϭ 6) cpm Ϯ S.E. of one representative experiment. A, expression of mouse WT GnRHRderived 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 Ϫ7 M Buserelin, and IP production was measured. Data are presented as the mean (n ϭ 6) cpm Ϯ 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 Ϯ 0.9 cpm (A) and 81.4 Ϯ 2.9 cpm (B-D).