| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,
,1
* Divisions of Neuroscience and Reproductive Sciences, Oregon National Primate Research Center and
Departments of Physiology and Pharmacology and
Cell and Developmental Biology, Oregon Health and Science University, Beaverton, Oregon, USA
1 Correspondence: ONPRC/OHSU, 505 NW 185th Ave., Beaverton, OR 97006, USA. E-mail: connm{at}ohsu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: quality control system • pharmacoperone • pharmacological chaperone • protein routing • receptor evolution
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
2
3
). These small molecules penetrate cells and correct folding, allowing mutants to escape detection/retention by the cell’s quality control system (QCS; refs. 4
5
6
).
By increasing the percentage of GnRHR molecules that route to the plasma membrane (PM), pharmacoperones have revealed that wild-type (WT) GnRHRs also engage the QCS, resulting in species-specific regulation. A substantial amount of newly synthesized WT protein may be retained/destroyed in the endoplasmic reticulum (ER; refs. 3
, 7
), as is the case for mutants (3
4
, 8
9
10)
, and this event appears to provide the underlying mechanism for a novel level of post-translational regulation (7
, 11)
of WT proteins. Retention of WT proteins is sometimes referred to as "inefficient" protein utilization (12)
. In the case of the GnRHR, a significant proportion of WT human, but not WT rat or mouse, GnRHRs are misfolded proteins, and are retained by the QCS and destroyed without ever reaching the PM (7
, 13)
.
Convergent evolution, occurring under strong pressure (7
, 11)
and utilizing several distinct strategies, diminishes net PM concentration of mammalian WT GnRHR. The GnRHRs of fish, reptiles, and birds have a long carboxyl-terminal that prolongs its half-life at the PM (14
15
16)
. This structure is lost among mammalian GnRHRs, which have no cytoplasmic extension at the carboxyl-terminal, unlike most other members of its GPCR superfamily. Among mammalian GnRHRs (usually containing 328 amino acids), there frequently is a Glu191 (among preprimates) or Lys191 (among primates).
This is true with the notable exception of rats and mice (containing 327 amino acids), which lack this "extra" amino acid and appear to route to the PM with higher efficiency than other mammals. In the human GnRHR, Lys191 is more effective than Glu191 at decreasing Bmax (7)
, although these amino acids each exert their effect by decreasing the probability of formation of the Cys14-Cys200 bond. Failure of this bond to form results in a GnRHR that is misfolded, recognized by the QCS, and retained in the ER (7)
. When comparing human and rat GnRHRs, which differ by 39 amino acids (and the presence of Lys191), a motif of four nonadjacent amino acids was identified in humans that accounts for the decreased expression of hGnRHR when Lys191 is present (7)
.
For the GnRHR, information is conveyed by both frequency and amplitude modulation. Graded receptor responses to amplitude modulation have been well studied in many systems and typically involve summation of quantal responses from individual cells with different set points. It remains unclear how receptors transduce frequency-modulated signals, however.
The GnRH ligand-receptor system has characteristics that appear to be associated with its role in reception of frequency-modulated signals. First, the pituitary gonadotrope cell that contains the GnRHR is linked to the hypothalamus by a very small volume and closed portal system; this allows delivery of a stimulus wave that has not been damped by dilution in the peripheral circulation. Second, the GnRH molecule itself is extremely labile (t1/2 is 
3 min in blood), so that the stimulus is not persistent. Little is known about specializations associated with the receptor, although it may be associated with levels of expression at the PM (i.e., the site of action) and affinity for the ligand. The Bmax and Ka regulate sensitivity of the response system as well as the ability to suppress (squelch) background noise.
The results in the present study identify specific amino acid residues of the GnRHR that vary among primates and compared with preprimate species. These appear to regulate the plasma membrane expression (PME) of GnRHR and the binding interaction with its ligand, two effects that potentially regulate the sensitivity of the response system and serve to squelch low-level noise signals. These effects appear to be specializations associated with reception of frequency-modulated signals.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 6
), myo-[2-3H(N)]-inositol and Na[125I] (Perkin Elmer, Boston, MA, USA; NET-114A and NEZ-033L, respectively). Dulbecco’s modified Eagle medium (DMEM), OPTI-MEM, lipofectamine, PBS (GIBCO, Invitrogen), competent cells (Promega, Madison, WI, USA), and Endofree maxi-prep kits (Qiagen, Valencia, CA, USA) were obtained as indicated and product purity was confirmed by optical density ratios following standard procedures recommended in the kit instructions.
Receptor mutagenesis
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 (Perkin Elmer, Foster City, CA, USA).
Transient transfection
Cells were cultured in growth medium (DMEM, 10% fetal calf serum (FCS), 20 µg/ml 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 growth medium in 48-well Costar cell culture plates. Twenty-four hours after plating the cells were washed once with 0.5 ml OPTI-MEM, then transfected with 5 or 25 ng/well of WT or mutant receptor, as indicated, with 95 or 75 ng pcDNA3.1 (empty vector) to keep the total amount of DNA at 100 ng/well. Lipofectamine was used according to the manufacturer’s instructions. Five hours after transfection, 0.125 ml DMEM with 20% FCS and 20 µg/ml gentamicin was added.
Twenty-three hours after transfection the medium was replaced with 0.25 ml fresh growth medium. Where indicated, 1 µg/ml IN3 in 1% DMSO (vehicle) was added for 4 h in media to the cells, then removed 18 h before agonist treatment (7)
.
Inositol phosphate assays
Twenty-seven hours after transfection, cells were washed twice with 0.5 ml DMEM/0.1% BSA/20 µg/ml gentamicin, preloaded for 18 h with 0.25 ml of 4 µCi/ml myo-[2-3H(N)]-inositol in inositol-free DMEM, then washed twice with 0.30 ml DMEM (inositol-free) containing 5 mM LiCl and treated for 2 h with 0.25 ml of a saturating concentration of Buserelin (10–6 M) in the same medium (or indicated amounts in dose-response curves). Buserelin (a GnRH agonist that contains both a D-amino acid6 and a Gly10 replaced by ethyamide) was used in these studies instead of the naturally occurring peptide, GnRH, because it is more stable in biological systems and binds to the receptor in the same pocket. Radiolabeled GnRH is not an acceptable radioligand for this receptor owing to its lability. Moreover, merely radiolabeling the molecule increases its affinity for the receptor by stabilizing a ß turn in the peptide backbone, and triturated peptide is too low in specific activity to be useful. Total inositol phosphate (IP) was determined (8)
. 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 (1
2
3
4
5
, 7
, 13)
.
Radioligand binding
Cells were cultured and plated in growth medium as described above, except 105 cells in 0.5 ml growth medium were added to 24-well Costar cell culture plates (cell transfection and medium volumes were doubled accordingly). Twenty-three hours after transfection, the medium was removed and replaced with 0.5 ml fresh growth medium. Twenty-seven hours after transfection, cells were washed twice with 0.5 ml DMEM containing 0.1% BSA and 20 µg/ml gentamicin, then 0.5 ml of DMEM was added. After 18 h, cells were washed twice with 0.5 ml DMEM/0.1% BSA/10 mM HEPES, then a range of concentrations of [125I]-Buserelin prepared in our laboratory (as modified from ref. 7
for use in cell cultures, specific activity is 700–800 µCi/µg, from 1.25x105 to 4x106 cam/ml, see nM in figure) in 0.5 ml of the same medium was added to the cells and allowed to incubate at room temperature for 90 min (7)
. This time was selected in order to achieve maximal binding. The amount of new receptor synthesis in this period is negligible at room temperature. After 90 min, the media was removed and radioactivity was measured as described previously (7)
. 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 (Bmax and Kd) were computed with Sigma Plot 8.02 (Handel Scientific Software, Chicago, IL, USA), using a nonlinear one-site binding model.
Statistics
Data (n
3) were analyzed with one-way ANOVA and the Student’s t test (Sigma Stat 3.1, Handel Scientific Software, Chicago, IL, USA; P<0.05 was considered significant).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
A is a schematic of the human GnRHR highlighting the positions of particular residues of significance in this study. Mammals other than rats and mice contain an extra amino acid at position 191 (Fig. 1A, B
). Frequently this is Glu191, although in primates it is a Lys191 (shown in red), a substitution that more effectively destabilizes a critical association of Cys14-Cys200 (orange), leading to bridge formation in hGnRHR (not required in rats, mice, or hGnRHR from which Lys191 is deleted) required for proper folding of the GnRHR (7)
. A motif consisting of amino acids 112/208/300/302 (shown in yellow, Fig. 1A, B
), which plays a role in allowing Cys14-Cys200 bridge destabilization by Lys191, is shown. Figure 1C
shows 11 amino acids that are heterogeneous among the four primate sequences examined in this study: two macaques (bonnet and rhesus), the chimpanzee, and the human. Rat sequence data are shown for reference since, in 9 of 11 circumstances, the residue found in rat is shared by one or more primates.
|
Unique modifications in the human and rhesus GnRHRs are associated with decreased affinity for the GnRH agonist
Figure 2
shows the expression and affinity of GnRH receptors from a broad range of species including the catfish, rat, mouse, and four primate species, the bonnet and rhesus macaques, chimpanzee, and human as well as some mutants. The catfish GnRHR has the lowest affinity of all species examined, followed by the primates (lowest affinity in the rhesus, followed by the human, bonnet macaque, and chimp, respectively), with rodents having the highest affinity. The catfish shows high capacity, followed by rodents. The binding capacity (Bmax) of the primate WTs is about the same and is the lowest of the WT sequences examined. Since the catfish GnRHR has such a low affinity for GnRH, saturation binding with this receptor was not achievable, and so is likely the source of the high degree of error observed between experiments with that receptor.
|
In the human sequence, conversion of Leu300 to Val is associated with a dramatic increase in ligand affinity (3-fold, P<0.05 compared with WT), suggesting that this amino acid may be a point of contact with GnRH agonist, yet has no effect on the Bmax. This observation indicates that altering the ligand binding affinity can occur without altering the trafficking pattern in the cell. When Lys191 was deleted from the hGnRHR (Fig. 2D
), the affinity for ligand also increased (P<0.05). Similarly, deletion of this amino acid from the human Leu300Val mutant was associated with an additional increase in affinity compared with the WT or mutant alone (P<0.05).
Mutation of Ser54Ala in the rhesus decreased the Kd (compared with rhesus WT GnRHR, P<0.05), butshowed no loss of Bmax (Fig. 2C, D
). Like the human substitution at position 300, this finding suggests that the affinity for ligand can be regulated independently of expression at the PM. Mutation of Ser54 to Ala increased the affinity of the mutant for ligand to near the human WT level (P<0.05).
Inositol phosphate production of GnRHRs from primates and comparative species
Figure 3
shows the efficacy and potency of G-protein coupling for the four primate receptors: two rodents and catfish receptors. These species can be considered in groups that reflect both species relations and relative responsiveness to the GnRH agonist, Buserelin. Rats and mice show higher efficacy and greater potency for Buserelin than do the four primates. This same data are shown as a percentage of maximum IP production (Fig. 3C
). EC50 and maximal IP production for each receptor are shown (Fig. 3C
).
|
Lys191 destabilizes a Cys14-Cys200 bridge in primates, but not in rats or mice
Figure 4
A shows the effect of removal of the Lys191 on maximum IP production of the WT receptor of the four primates examined or its addition to WT rat and mouse sequences (which normally do not contain it). Note that the values in Fig. 4
have been normalized to account for different expression levels between species. The WT rat and mouse GnRHRs are minimally, if at all, affected by its addition, since these species also lack the motif that allows its action (7)
. Breaking the bridge between the amino-terminal and ECL2 (by mutation of Cys14 to Ala) did not significantly change the level of expression of rat or mouse receptors at the PM, but decreased expression among the primates, an event that was reversed by deletion of Lys191 (7)
.
|
All primate GnRHRs are inefficiently expressed
Figure 4B
shows the effect of pharmacoperone IN3 on WT human GnRHR. Increased IP production of the WT receptor after treatment with the pharmacoperone or after deletion of Lys191 is due to characteristic inefficient expression of this WT receptor (7)
, since a portion is ordinarily misfolded, retained, and degraded in the ER. The percentage of hGnRHR that is correctly folded can be increased by the use of pharmacoperone or by deleting the Lys191
The homologous exchange of Leu for Val at position 300, along with the complementary exchange of Val for Leu at position 155 (Fig. 1C
; indicated by pound symbols), are the only changes that distinguish the human from all other primates. Amino acid 300 is part of a motif associated with the ability of Lys191 to destabilize the Cys14-Cys200 bridge in human GnRHR (7)
.
Accordingly, we created hGnRHR(Leu300Val) and observed that this modification has little effect on the maximum level of response to GnRH agonist (compared with WT) even when Lys191 is deleted (compared with the template of WT with the Lys191 deleted; Fig. 2
and Fig. 4B
). When the Cys14-Cys200 bridge of WT or Leu300Val human GnRHR is also broken by conversion of the Cys14 to Ala, this results in loss of activity that is restored by deletion of Lys191 (Fig. 4B
). These latter results suggest that the loss of activity is likely due to ER retention of a misfolded protein.
In comparing sequences among the primates, we also noticed a Ser54 that, in primates, is unique to the rhesus, although seen in some preprimates such as horse, sheep, pigs, guinea pigs, mice, rats, and opossums. Like Leu300Val human GnRHR, this modification did not change the receptor efficacy (compared with rhesus WT).
Interaction between position 217, modifications at 225, and the 112/208/300/302 motif
We have shown that transmembrane segment 4 (TMS4) and TMS5 contain amino acids that can bend the protein backbone leading to altered geometry between the amino-terminal and the ECL2 and alignment of the Cys residues involved in formation of the Cys14-Cys200 bridge (7)
. Accordingly, we prepared mutants of the human receptor that contained Gly217 and rodent comparators with Ser216, since this amino acid has been implicated in regulatory differences (13)
.
Making the mouse "rat-like" at position 216 (the position homologous to human 217) resulted in a modest destabilizing effect when Lys191 was inserted (compared with the mouse Gly216Ser lacking Lys191, P<0.05; Fig. 5
A–C), suggesting a modest requirement for the Cys14-Cys200 bridge compared with mouse WT GnRHR (13)
. Making the rat "mouse-like" at this position had little effect on IP production compared with WT rat GnRHR; neither the Ser216Gly nor Ser216Gly/Cys14Ala showed a significant inhibition of Lys191. The rat Ser216Gly/Cys14Ala produced IP at slightly reduced levels compared with either WT (P<0.05) or Ser216Gly (P<0.05), suggesting that the Gly in the mouse WT provides flexibility and does not provide the structure needed for the role of Lys191 as a bridge destabilizer.
|
In the human GnRHR, conversion of Ser217 to Gly (Fig. 5A
) results in loss of IP production in response to a saturating concentration of agonist (P<0.05), but this can be compensated for by deletion of Lys191. Thus, it is reasonable to believe that in the human structure, Ser217 stabilizes a bend in the peptide backbone that aligns the amino acids necessary to form the Cys14-Cys200 bridge. These observations are consistent with a view that, in the human, Ser217 formalizes a turn in TMS5 that helps to align the Cys14-Cys200 bridge (7
, 13)
. In the human Ser217Gly mutant, when the Cys14-Cys200 bridge was broken by conversion of Cys14 to Ala, the result was an almost complete loss of IP production in response to agonist. Loss of activity can be rescued by deletion of Lys191; these effects are virtually identical to what is observed for WT human GnRHR in which the bridge is broken and the Lys191 deleted (7)
.
Adding flexibility in the human sequence by converting Ser217Gly results in loss of IP production in response to agonist in the WT sequence, but increases it when the rat 112/208/300/302 motif is present (P<0.05; Fig. 5E
). When Cys14 is converted to Ala in the presence of Gly217 in a mutant also containing the rat 112/208/300/302 motif, IP production in response to agonist is similar to that observed by WT human GnRHR (Fig. 5F
). This suggests that the human 112/208/300/302 motif is required for the action of Ser217 on stabilization of the relation of the Cys14 and Cys200 needed for bridge formation.
We also examined position 225 (Fig. 5D-G
). Of the species examined in this study, Phe225 is unique to the chimpanzee and human. Even among all other mammals cloned to date, only the dog GnRHR contains Phe225; the remaining species contain Leu in the homologous position. The dog (with Phe225) also features a highly flexible Gly168 that is a position associated with torsion of the extracellular loop 2 (ECL2, 7). This added flexibility may be the means by which the dog receptor tolerates the Phe225.
Modification of the human GnRHR Phe225 to Leu results in loss of IP production compared with WT receptor and decreases the impact of removal of Lys191 (P<0.05; Fig. 5D
). The human sequence has progressively rigidified both Ser217 and Phe225, presumably to more rigorously control the formation of receptor folding and routing to the PM, likely by decreasing the probability of formation of the Cys14-Cys200 bridge (Fig. 5F
). The Phe225Leu modification has an effect similar (Fig. 5D, E
) to that of Ser217 by decreasing IP production compared with the WT sequence and increasing it when the human amino acids at positions 112, 208, 300, and 302 are replaced by the rat motif (Fig. 5E
). When the Cys14-Cys200 bridge is broken (Fig. 5F
) and the rat motif is present, the Phe225Leu modification enhanced IP production in response to agonist compared with the WT receptor. When the bridge is intact (Fig. 5G
), a combination of the Phe225Leu modification with either Leu112Phe, Gln208Glu, or Leu300Val/Asp302Glu alone was less effective at increasing IP production than when the entire 112/208/300/302 motif was changed to the amino acids found in the rat.
These results identify specific amino acid residues of the GnRHR that vary among primates compared with preprimate species and have the ability to regulate levels of PME and ligand binding affinity.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; GnRH plasticity is a general feature of mammals, although the details of specific distinctions continue to be the target of active study (18
, 19)
. Accordingly, accurate sensing of pulses is important for ovulation, an event less rigorously controlled in rats, animals with a shorter cycle (20)
.
Amino acids associated with control of PME
The present study uses radioligand binding and effector activation in response to agonist occupancy to examine the primate GnRH receptor in light of a motif (amino acids 112/208/300/302) that participates in regulation of Bmax in the human. In the 20 years since the sequence of this receptor was reported, no laboratory has succeeded in producing an antiserum that has been rigorously shown to recognize the GnRHR, so blots cannot be performed.
Regulation of the PME of the GnRHR occurs by allowing Lys191 to destabilize the Cys14-Cys200 bridge, a critical determinant for primate GnRHRs to pass the QCS of the cell (7)
. Failure to form this bridge (in primates) results in production of a misfolded mutant rescue with a pharmacoperone or by deletion of the primate Lys191. Replacement of the human GnRHR residues in this motif by the rat ortholog both increases net expression and decreases the need for the Cys14-Cys200 bridge to form (as assessed by the Cys14Ala mutants; see ref. 7
).
Our data also reveal a role for amino acid 217 in the human GnRHR. This is a significant site since Ser217Arg is one of only two naturally occurring mutants that cannot be rescued with different pharmacoperones (5)
. It has become apparent that this thermodynamically unfavorable substitution results in a mutant in which the Cys14-Cys200 bridge can never form and is retained in the ER (7)
. Because ECL2 connects TMS4 and TMS5, it is likely that this mutant affects the spatial relation needed for the Cys14-Cys200 bond (between the amino-terminal region and ECL2). The orthologous amino acid in rodents is also the underlying basis of the difference in PME and dominant-negative actions (6
, 10)
in the rat and mouse (13)
. The dominant-negative action occurs when mutant receptors retain the WT in the ER as a result of oligomerization.
The human Ser217 may formalize a turn, allowing an alignment of Cys14 and Cys200. Ser with a slightly polar nature, small size, and a propensity of the side-chain hydroxyl oxygen to hydrogen bond with the protein backbone, frequently found in association with tight turns of protein structure. In contrast, Gly is racemic and highly flexible. Accordingly, the human sequence has progressively rigidified position 217 in order to more rigorously control expression at the PM, likely by regulating the probability of formation of the Cys14-Cys200 bridge. When this bridge is intact, the combination of the Phe225Leu modification with Leu112Phe, Gln208Glu, or Leu300Val/Asp302Glu alone was less effective at increasing IP production than when the entire 112/208/300/302 motif was changed to the amino acids found in the rat.
Amino acid positions associated with control of ligand binding affinity
We also describe amino acid substitutions (positions 54 and 300 in the rhesus and human, respectively) that affect the binding affinity without a substantial effect on the Bmax of the protein expressed. Because of the position of the amino acid 300 in the molecule, it is easier to imagine that a change at this position (ECL3) alters affinity by direct interaction with the ligand, although this appears close to a binding pocket (21)
. This case is harder to make with position 54 located near the cytoplasmic side of the first TMS (Fig. 1A
), although other residues buried in the lipid bilayer appear accessible to ligands (22)
, and biologically active GnRH analogs are hydrophobic. A change in affinity from a modification at this site may also be a tribute to the interactive nature of the seven transmembrane structure. Modification of WT rhesus Ser54Ala increases affinity to the same level as the closely related bonnet and human, thus compensating for the only other two variances in structure: bonnet has Leu6 (rather than Ser6) and Lys248 (instead of Glu248). Accordingly, the WT human and WT rhesus have markedly decreased affinity for the ligand compared with the mouse or rat. The WT bonnet GnRHR is similar in ligand binding affinity to the human, but the rhesus has the poorest affinity of all primates. Lys191 found in primate WT GnRHRs also decreases binding affinity, although this is accompanied by a decrease in Bmax, an effect previously reported for a chimera of this sequence with the carboxyl tail of the catfish (23)
. The fact that nature has relied on changes at two different residues (in different primates) to achieve a decrease in affinity is a tribute to the convergent pressure for this event.
Ligand binding affinity as a noise suppression regulator (squelch control)
The likely selective advantage of decreased affinity is clearer when one considers that the GnRHR in primates is governed by ligand frequency modulation, making the distinction of individual pulses important. The dilution of any square wave into a liquid will result in blunting into a sinusoidal wave pattern with alternating low, high, and intermediate levels of GnRH rather than the "completely on" or "completely off" pattern characteristic of a square wave. In principle, a completely off phase may never occur at times of high pulsatility. Accordingly, decreasing the binding affinity is an effective strategy for ignoring low-level stimuli, effectively squelching (suppressing) noise in the system, an advantage in a frequency-modulated system.
Inefficient PME (12)
occurs for the primate GnRHR and may be a "mass action" regulatory mechanism used by proteins as well (24
25
26
, 22)
.
The observation that it has evolved (in the GnRHR) under strong convergent pressure and despite the metabolic waste of unused receptor, along with added susceptibility to mutational disease (13)
, leads to the conclusion that it must have value. One attractive possibility is that post-translational regulation might involve control by endogenous protein chaperones, which may themselves be regulated by the steroidal milieu. In this manner they might participate in the oscillation of the GnRHR through the reproductive cycle.
Restriction of the PME of the GnRHR appears concurrently with the increased regulatory control of reproduction that occurs in going from premammals (birds, fish, reptiles; animals that produce large numbers of eggs/offspring with a low metabolic investment in each and low survival rates) to mammals, including primates (animals with greater metabolic investment and higher survival rates; ref. 7
).
Receptor concentration at the PM as a gain control
The sensitivity of the human GnRHR to balance between the PM and ER is seen by the exquisite sensitivity to charge changes. The majority of mutations causing HH, 11 of the 17 reported mutants, involve change of charge of single amino acids (9)
. Even simple changes in hydrophobicity appear to be associated with needed compensating changes. Among primates, for example, conversion of Phe112 (rhesus, bonnet, rat) to Leu112 (human, chimp) is accompanied by a reverse conversion of Leu224/225 (rhesus, bonnet, rat) to Phe224/225 (human, chimp; Fig. 1C
, asterisks). Likewise, conversion of the human to Leu300 (chimps, rhesus, bonnet, and rat are Val299/300) is accompanied by a complementary conversion at position 155 (Val155 in the human and Leu155 in chimp, bonnet, rhesus, and rat; Fig. 1C
, pound symbols).
Frequency modulation in signaling systems
The present data show modifications of the primate GnRHR associated with decreased ligand binding affinity. These modifications appear to have evolved to provide a "squelching" system that enhances the ability to distinguish pulse frequency changes from background noise. Other changes in primates continue to play on an evolutionary theme in which Bmax is progressively decreased, a mechanism capable of regulating receptor numbers in response to altered frequency of stimulation, as a homeostatic mechanism. Together, these modifications provide specializations appropriate for detection of a frequency-modulated signal, enabling one stimulus to regulate multiple responses. The present observations will be valuable for understanding the means by which pulsatile signals are transduced, since many frequency-modulated systems have been reported recently and aberrancies are often an underlying cause of disease (27
28
29
30
31
32
33
34
35)
.
|
ACKNOWLEDGMENTS |
|---|
Received for publication July 31, 2006. Accepted for publication September 14, 2006.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
optoid receptor maturation. EMBO J. 21,1628-1637[CrossRef][Medline]This article has been cited by other articles:
|
|
 |
|
  E. Jardon-Valadez, A. Aguilar-Rojas, G. Maya-Nunez, A. Leanos-Miranda, A. Pineiro, P M. Conn, and A. Ulloa-Aguirre Conformational effects of Lys191 in the human GnRH receptor: mutagenesis and molecular dynamics simulations studies J. Endocrinol., May 1, 2009; 201(2): 297 - 307. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. M. Conn, A. Ulloa-Aguirre, J. Ito, and J. A. Janovick G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo Pharmacol. Rev., September 1, 2007; 59(3): 225 - 250. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. W. Szkudlinski Challenges and Opportunities of Trapping Ligands Mol. Pharmacol., August 1, 2007; 72(2): 231 - 234. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
