A Ligand-Mimetic Model for Constitutive Activation of the Melanocortin-1 Receptor

Dongsi Lu, Dag Inge Vage and Roger D. Cone

Vollum Institute (D.L., R.D.C.) Oregon Health Sciences University Portland, Oregon 97201
Department of Animal Science (D.I.V.) Agricultural University of Norway N-1432 As, Norway


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Dark coat color in the mouse and fox results from constitutively activated melanocortin-1 receptors. Receptor mutations in the mouse (E92K, L98P), cow (L99P), fox (C125R), and sheep (D119N) cluster near the membrane/extracellular junctions of the second and third transmembrane domains, an acidic domain that is the likely site of electrostatic interaction with an arginine residue in the ligand, {alpha}-MSH. For transmembrane residues E92, D119, and C125, conversion to a basic residue is required for constitutive activation. Unlike constitutively activating mutations in many G protein-coupled receptors that increase agonist efficacy and affinity, these MC1-R mutations have the opposite effect. Therefore, these mutations do not activate the receptor by directly disrupting intramolecular constraints on formation of the active high-affinity state, R*, but do so indirectly by mimicking ligand binding.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The MSH receptor (MSH-R or MC1-R) regulates the eumelanin/phaeomelanin switch in melanocytes, controlling the relative amount of yellow-red phaeomelanin and brown-black eumelanin pigments ultimately deposited in skin and hair (5). The MC1-R couples through Gs to adenylyl cyclase to stimulate tyrosinase, the rate-limiting enzyme in the synthesis of both classes of melanin pigments (13, 14). For reasons that are not yet understood, basal levels of tyrosinase expression lead largely to phaeomelanin synthesis while elevated levels of tyrosinase, resulting from {alpha}-MSH stimulation of melanocytes, divert intermediates primarily along the eumelanin synthetic pathway (12, 13, 14).

Two genetic loci known to regulate the eumelanin/phaeomelanin switch are agouti and extension, and most domesticated animals have multiple alleles at each of these loci (36). The agouti and extension alleles in concert determine the distribution of phaeomelanin and eumelanin, both along each hair shaft as well as spatially along the coat of the animal. Dominant alleles at extension result in dark brown or black coat color while animals homozygous for recessive alleles have yellow or red coats; the opposite is true for agouti alleles. The identification of the MC1-R as the extension locus in the mouse led to the finding that a point mutation, E92K, in the dominant Eso-3J allele resulted in a receptor that was constitutively active, defined as being able to significantly elevate adenylyl cyclase activity in the absence of ligand (28). A second allele (Eso, L98P) was found in an independent occurrence of the Sombre phenotype and also produces a constitutively active receptor (5). More recently, a second constitutively active MC1-R has been characterized in the red fox, Vulpes vulpes (42). In this animal, a C125R change in the third membrane-spanning domain produces the dark black morph, known as the Alaska silver fox. Additional dominant mutations in the MC1-R have now been reported in cattle (17) and in chickens (38); however, these mutations have not yet been characterized pharmacologically.

Constitutively active G protein-coupled receptors (GPCRs) were first identified in chimeras of the {alpha}1- and ß2-adrenergic receptors (6). Ultimately, this effect was mapped to residues at the C-terminal end of the third intracellular loop, and, in particular, the replacement of an alanine at position 293 with any other residue was found to increase the basal activity of the receptor and enhance the affinity for ligand as much as 100 fold (16). After the identification of naturally occurring constitutively active MC1-Rs (28) and rhodopsin molecules (29), activating mutations in GPCRs were found to be responsible for a diverse array of inherited as well as somatic genetic disorders including hyperfunctioning thyroid adenomas (21, 23, 43), autosomal dominant hyperthyroidism (40, 41, 44), familial precocious male puberty (18, 37, 45), metaphyseal chondrodysplasia (35), familial hypoparathyroidism (22, 39), and congenital night blindness (8, 25).

While constitutively activating mutations have been found in virtually all domains of the GPCRs, some mechanistic similarities are commonly found. Many constitutively active receptors demonstrate a higher affinity for agonist and lower EC50 for further activation (37, 43). In some cases the increased affinity for agonist, but not antagonist, was dramatic (27), and the correlation between agonist efficacy and increased affinity in the constitutively active mutants (31) led to a proposed modification of the ternary complex model for GPCR activation (7). The established model holds that agonist binding stabilizes the active conformation (R*G) of the receptor in a complex with G protein while antagonists typically bind equally well to R and R*. Based on the identification and characterization of constitutively active GPCRs, an extended or allosteric ternary complex model was proposed in which receptor, independent of ligand binding, is in equilibrium between an inactive and active conformation (19). Mutations that constitutively activate receptors are proposed to disrupt internal constraints in the receptors, make the receptors less conformationally constrained (10), and therefore decrease the energy required to reach the R* state. The model thus explains the increased affinity of agonists for constitutively active receptors, even in the absence of G protein, since constitutive activation results in a higher percentage of receptors in the high-affinity R* state.

Two constitutively activating mutations of the mMC1-R, an E92K change near the extracellular margin of the second transmembrane domain and a L98P change approximately two turns in the {alpha}-helix above E92, were initially predicted to activate the receptor by directly disrupting an internal constraint resulting from an electrostatic interaction between E92 and another unidentified residue (28). The L98P was predicted to alter the constraint involving E92 by disrupting the {alpha}-helical structure of the second transmembrane domain in which E92 was found. A similar model was proposed for a K296E mutation found in the seventh membrane-spanning domain of rhodopsin in a family with retinitis pigmentosa (29). This mutation, which occurs at the site of covalent attachment of the retinal chromophore, was found to produce an opsin that was capable of fully activating transducin in the absence of light or retinal. Twelve different amino acid substitutions at this position have been examined, and all but the basic lysine or arginine residues constitutively activate the receptor (4). Furthermore, a glutamic acid at position 113 is generally accepted as a counterion of the Schiff base linkage between K296 and retinal (20, 30), and mutagenesis of this glutamic acid residue to a Gln also constitutively activates rhodopsin (29).

Remarkably, however, in contradiction to the allosteric ternary complex model, the E92K, L98P, and C125R mutations constitutively activate the MC1-R but dramatically lower agonist affinity and efficacy. We show here that these mutations do not directly disrupt internal constraints favoring the inactive receptor conformation. Rather, these constitutively activating mutations cluster in an acidic domain of the receptor predicted to interact electrostatically with an arginine residue in the agonist that is required for high-affinity binding. We demonstrate that introduction of a basic residue in this region of the receptor is essential for constitutive activation and propose that these mutations activate the receptor by ligand mimicry.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Insertion of a Basic Residue at Position 92 Is Required for Constitutive Activation of the Mouse Sombre Allele of the MC1-R (Eso-3J)
To understand the mechanism of constitutive activation of the MC1-R, in vitro mutagenesis was performed at a number of positions in the receptor identified as activating positions in the mouse (E92, L98P), fox (C125), and sheep (D119), and at a conserved position, D115, in this same domain (Fig. 1Go). Agonist efficacy was determined using the native peptide ligand, {alpha}-MSH, and a synthetic ligand, NDP-{alpha}-MSH, which has a higher affinity for receptor (32). Throughout this study, a colorimetric cAMP-dependent ß-galactosidase assay was used for analysis of the level of activity of the Gs-coupled MC1-R; previous studies demonstrate this assay provides results comparable to conventional adenylyl cyclase assays (2, 28).



View larger version (70K):
[in this window]
[in a new window]
 
Figure 1. Coat Color Phenotypes in Animals with Wild-Type or Dominant Extension Allele(s) Resulting from Amino Acid Variants of the MC1-R

A, Amino acid variants of the MC1-R. Naturally occurring dominant mutations from different species are indicated with arrows, and the species involved and extension alleles associated with these mutations are indicated. The positions of these residues are shown at the homologous positions in the mouse MC1-R. Asterisks indicate the positions of all residues examined in this study. B, Coat colors of mice with different extension alleles. A mouse with wild-type extension alleles E is on the left, and a sombre mouse with dominant extension allele Esom-3J is on the right. C, Coat colors of cattle with different extension alleles. Cattle with red coat color have recessive extension alleles e/e, and cattle with dark coat color have the dominant extension allele ED. D, Coat colors in the red fox and silver fox. A red fox with wild-type extension alleles E/E is on the top, and a silver fox with a dominant extension allele EA is on the bottom. E, Coat colors of sheep with different extension alleles. The sheep with white coat color has wild-type extension alleles, and the sheep with dark coat color has a dominant extension allele.

 
Glutamic acid 92 of the murine MC1-R was mutated to alanine, aspartate, lysine, glutamine, and arginine to determine requirements at this position for constitutive activation. All changes in position 92 increased the EC50 for activation of the mutant receptors by either {alpha}-MSH (Fig. 2AGo and Table 1Go) or NDP-{alpha}-MSH (Fig. 2BGo and Table 1Go). Only introduction of a basic lysine or arginine at this position resulted in constitutively active receptors. Both receptors containing basic residues at position 92 showed elevated basal cAMP-responsive ß-galactosidase activity, even in the absence of hormone stimulation. The basal level of the E92K mutant receptor was about 30% to 50% of the maximal stimulation level of the wild-type receptor achieved by treatment with 10-6 M {alpha}-MSH or [norleucine4,D-phenylalanine7]-{alpha}-MSH (NDP-{alpha}-MSH). However, the E92K mutant receptor could be fully activated by higher concentrations of either {alpha}-MSH or NDP-{alpha}-MSH to levels comparable to maximal activation of the wild-type receptor. The basal activity of the E92R mutant was about 60% to 90% of the maximal stimulation level of the wild-type receptor.



View larger version (40K):
[in this window]
[in a new window]
 
Figure 2. Pharmacology of the E92 Mutants and Proline Mutants of the Mouse MC1-R

A, {alpha}-MSH stimulation curves for the mouse MC1-R wild-type and E92 mutants. Basic residue changes E92K and E92R result in constitutive activation of the receptor. Wild-type and mutant MC1 receptors are assayed by analyzing their ability to activate expression of a cAMP-induced ß-galactosidase fusion gene. Cells stably expressing each receptor and transiently expressing the fusion construct were stimulated for 6 h with medium alone, 10 µM forskolin, or increasing concentrations of {alpha}-MSH (A), or NDP-{alpha}-MSH (B and E), after which ß-galactosidase concentrations were determined. Data points represent means of triplicate determinations divided by maximal levels of ß-galactosidase activity achieved by 10 µM forskolin stimulation to normalize for transfection efficiency, and error bars indicate SDs. B, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and E92 mutants. Superpotent ligand NDP-{alpha}-MSH further activates the E92K mutant to levels equivalent to maximal stimulation of the wild-type receptor by 10-6 M NDP-{alpha}-MSH. Methods are as in panel A, above. C, Competition binding curves for the mouse MC1-R wild-type and E92 mutants. In contrast to an alanine change at the E92 position, basic residue change E92K results in reduced ligand affinity. Increasing concentrations of cold NDP-{alpha}-MSH are used to compete with the [125I]NDP-{alpha}-MSH tracer. Nonspecific binding is determined as the counts bound in the presence of 10-5 M cold NDP-{alpha}-MSH. Data are displayed as percentage of maximal specific counts bound. Data points represent means of duplicate determinations, and bars indicate SDs. D, Scatchard analysis shows that the mouse E92K mutant of the MC1-R has 10-fold higher Kd value than the wild-type receptor, indicating the reduced binding affinity of the mutant MC1-R for ligand. Kd values of E92K and wild- type mMC1-R are 6.41 ± 2.52 nM, and 0.62 ± 0.30 nM, respectively. Inset shows the enlarged Scatchard curve for E92K mutant of the MC1-R. E, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and proline mutants. L98P mutant shows constitutive activation. Methods are as in panel A, above. F, Competition binding curves for the mouse MC1-R wild-type and proline mutants. L98P mutant shows reduced ligand affinity, while E100P mutant shows similar ligand affinity as the wild-type receptor. Increasing concentrations of cold NDP-{alpha}-MSH and cold {alpha}-MSH are used to compete with the [125I]NDP-{alpha}-MSH for E100P and L98P, respectively. Methods are as in panel C, above.

 

View this table:
[in this window]
[in a new window]
 
Table 1. EC50 Values for {alpha}-MSH and NDP-{alpha}-MSH Stimulation, Total Specific Binding, and IC50 Values of the mMC1-R Wild-Type and Mutants in Stably Transfected HEK 293 Cells

 
Competition binding studies (Fig. 2CGo and Table 1Go) demonstrated that IC50 values for the E92A (1.07 ± 0.53 nM) mutant receptor remained similar to the wild type (0.79 ± 0.48 nM), demonstrating that the glutamic acid residue is not directly involved in ligand binding. However, IC50 values for the E92K and E92Q mutants were increased to 13.7 ± 17.7 nM and 3230 ± 1930 nM, respectively. Binding to the E92R mutant receptor could not be detected. Mutations at the E92 position all dramatically lowered the amount of ligand binding detected in competition studies (Table 1Go), whether as a consequence of a reduction in receptor affinity (E92K), receptor number (E92A), or perhaps both. Scatchard analysis of the E92K mutant (Fig. 2DGo) supported the competition binding data and indicated a 10-fold increase in dissociation constant (Kd) value for this receptor (6.41 ± 2.52 nM vs. 0.62 ± 0.3 nM for the wild-type receptor), with little change in the number of binding sites.

Insertion of a Proline Residue Adjacent to E92 Constitutively Activates the MC1-R in Cattle (ED) and in an Independent Occurrence of the Sombre Mouse (Eso)
Two proline insertion events, L98P in an independent occurrence of the Sombre phenotype in mice (28), and L99P in black Icelandic cattle (17) have been found to be linked to dominant MC1-R alleles. Preliminary data demonstrated that the murine L98P receptor was constitutively active (5). The L98P receptor has a basal activity equal to 30% to 50% of the maximal stimulation level of the wild-type receptor (Fig. 2EGo and Table 1Go). The L98P mutant receptor can be slightly activated by {alpha}-MSH (data not shown) and nearly fully activated by NDP-{alpha}-MSH (Fig. 2EGo). Similar to the E92K and E92R mutants, the L98P mutant demonstrates constitutive activity and an increase in EC50 for stimulation by NDP-{alpha}-MSH from 0.016 ± 0.005 nM to 3.27 ± 0.19 nM. The IC50 for the L98P mutant receptor (301 ± 55 nM) was 100-fold higher than the wild-type receptor (Fig. 2FGo and Table 1Go).

While the L99P change in the bovine receptor was not characterized pharmacologically, it might be expected to behave similarly to the murine L98P mutation. To test the limits of the domain in which a proline insertion will constitutively activate the receptor, an E100P mutation was also constructed and tested pharmacologically. This mutant was found to be very similar to the wild-type receptor, with comparable EC50 and IC50 values (Fig. 2Go, E–F, and Table 1Go).

Mutation of Conserved Basic Receptor Residues H183, H258, and K276 Does Not Constitutively Activate the MC1-R
A hypothesis for constitutive activation of rhodopsin is that an ionic bridge between K296 and E113 constrains the receptor in an inactive conformation, since removal of the charge at either position will activate the receptor in the absence of light or retinal (3, 29). The requirement of a basic residue at position 92 for constitutive activation, rather than simple ablation of the negative charge, argues against a similar model for the MC1-R. Nevertheless, to test for basic residues that might stabilize the receptor via an electrostatic interaction with E92, we mutagenized all three conserved basic residues that might be in a position to interact with E92. In theory, disruption of such a residue should also result in a constitutively active receptor.

H258 and K276 were mutagenized to acidic residues, as well as to other amino acids; however, no significant constitutive activation resulted (Fig. 3Go). Similar data were seen for H183 (data not shown). Interestingly, both the H258E and K276 mutations increased maximal hormone-stimulated activity of the receptor. The EC50 for NDP-{alpha}-MSH stimulation was unchanged for the receptor with a H258E change. Introduction of isoleucine or tryptophan at this position increased the EC50 value for NDP-{alpha}-MSH stimulation by 5- and 20-fold, respectively, and made the receptor nearly resistant to {alpha}-MSH stimulation (data not shown). All three mutations at position 258 increased the IC50 for competition binding of {alpha}-MSH by 100-fold or more (Fig. 3BGo and Table 1Go). Quite differently, the H183E and K276E mutant receptors showed wild-type activation and binding properties. The two other K276 mutant receptors, K276A and K276L, showed about a 10-fold increase in EC50 for {alpha}-MSH stimulation but no change in the EC50 for NDP-{alpha}-MSH and no change in the IC50 (Table 1Go). Thus, removal of potentially basic residues at positions 183, 258, and 276 not only does not activate the receptor, but suggests that residues 183 and 276 have no major role in ligand binding or activation.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 3. Pharmacology of the H258, K276, and H183 Mutants of the mMC1-R

A, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and H258 mutants. All three H258 mutants have activation profiles similar to the wild- type receptor. Methods are as in Fig. 2AGo. B, Competition binding curves for the mouse MC1-R wild-type and histidine mutants. All three mutants at H258 greatly reduce ligand affinity, indicating that this residue is likely involved in ligand binding, while H183E shows similar affinity for the ligand as the wild-type receptor. Methods are as in Fig. 2CGo. C, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and K276 mutants. All three K276 mutants have activation profiles similar to the wild-type receptor. Methods are as in Fig. 2AGo. D, Competition binding curves for the mouse MC1-R wild-type and K276 mutants. All K276 mutants show similar affinity for the ligand as the wild- type receptor. Methods are as in Fig. 2CGo.

 
Insertion of a Basic Residue at Position 123 Is Required for Constitutive Activation of the Alaskan Silver Fox Allele (EA)
Recently, a mutation of a conserved cysteine at position 125 (equivalent to C123 in the mouse) to an arginine has been found to be associated with a dominant extension locus allele responsible for dark coat color seen in the Alaska silver fox (42). This mutation is capable of constitutively activating the mouse MC1-R to 80% maximal wild-type receptor levels in some experiments (42). The C123 residue is found in the middle of transmembrane domain 3 and was analyzed in a similar fashion to the E92 position. This residue was mutagenized from a noncharged polar residue to a nonpolar residue of similar size (alanine), an acidic residue (glutamate), and two basic residues (lysine and arginine). In parallel with the E92 position, only the insertion of a lysine or arginine at this position was found to constitutively activate the receptor (Fig. 4Go). C123K and C123R changes both constitutively activated the receptor to about 30–40% of the maximal stimulation level (mutant vs. wild-type basal activity: P < 0.0005, t test). These four mutations have wild-type IC50s for ligand binding (Fig. 4CGo and Table 1Go), indicating that C123 is not directly involved in ligand binding. Since the C123A change had no effect on ligand binding or activation of the receptor, it can be inferred that the disulfide-bonding capability of the cysteine plays no detectable role in ligand binding or receptor activation. Interestingly, this position behaved very similarly to the E92 position, in that constitutive activation resulting from the insertion of a basic residue greatly decreased the effectiveness of both {alpha}-MSH (Fig. 4AGo) and NDP-{alpha}-MSH (Fig. 4BGo) in further activation of the receptor.



View larger version (22K):
[in this window]
[in a new window]
 
Figure 4. Pharmacology of the C123 Mutants of the mMC1-R

A, {alpha}-MSH stimulation curves for the mouse MC1-R wild-type and C123 mutants. Basic residue changes C123R and C123K result in constitutive activation of the receptor. Methods are as in Fig. 2AGo. B, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and C123 mutants. The C123K and C123R mutants demonstrated elevated basal activity, and C123K could be further activated by the superpotent ligand NDP-{alpha}-MSH. Methods are as in Fig. 2AGo. C, Competition binding curves for the mouse MC1-R wild-type and C123 mutants. All C123 mutants have similar affinity for the ligand as the wild-type receptor. Methods are as in Fig. 2CGo.

 
Mutagenesis of Either of Two Closely Spaced Aspartates (D115 and D119) Leads to Constitutive Activation, with the Latter Change Being Associated with a Dominant Extension Allele in the Sheep (ED)
Independent of any genetic data implying a role in constitutive activation, in vitro mutagenesis studies were carried out on two aspartate residues, D115 and D119, located near the junction between the presumed first extracellular loop and transmembrane domain 3. With E92 apparently not involved in ligand binding, these two residues are the only other conserved acidic residues in a position to interact electrostatically with the arginine residue in the H-F-R-W pharmacophore of ligand {alpha}-MSH. Midway through the study, it was learned that the D119N change is associated, together with a M71K change, with a dominant extension locus allele in the sheep (D. I. Vage, unpublished data). The D119 position was mutagenized to three different amino acids: lysine, asparagine, and valine.

In parallel with the E92 and C123 positions, elevated basal level of cAMP-response ß-galactosidase activity equivalent to about 20–40% of the maximal stimulation level of the mMC1-R was only seen after the insertion of a basic residue at this position (Fig. 5AGo). Likewise, neither one of the three mutant receptors could be activated by {alpha}-MSH, while all three were activated by NDP-{alpha}-MSH at greatly reduced efficacy (Fig. 5BGo and Table 1Go). In contrast with changes at E92 and C123, all three D119 variants demonstrated a 10- to 100-fold increase in IC50 values for competition of NDP-{alpha}-MSH binding in comparison with the wild-type receptor, implying a contribution of D119 in high-affinity ligand binding (Fig. 5CGo and Table 1Go).



View larger version (44K):
[in this window]
[in a new window]
 
Figure 5. Pharmacology of the D119 and D115 Mutants of the mMC1-R

A, {alpha}-MSH stimulation curves for the mouse MC1-R wild-type and D119 mutants. Basic residue change D119K results in constitutive activation of the receptor. Methods are as in Fig. 2AGo. B, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and D119 mutants. All D119 mutants were activated by the superpotent ligand NDP-{alpha}-MSH with greatly reduced efficacy. Methods are as in Fig. 2AGo. C, Competition binding curves for the mouse MC1-R wild-type and D119 mutants. All D119 mutants show greatly reduced ligand affinity, indicating that this position is likely involved in high-affinity ligand binding. Methods are as in Fig. 2CGo. D, {alpha}-MSH stimulation curves for the mouse MC1-R wild-type and D115 mutants. All D115 mutant receptors are constitutively active. Methods are as in Fig. 2AGo. E, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and D115 mutants. Methods are as in Fig. 2AGo. F, Competition binding curves for the mouse MC1-R wild-type and D115 mutants. Unlike D115E and D115V, D115K results in reduced affinity for the ligand. Methods are as in Fig. 2CGo.

 
The D115 was mutagenized to three different amino acids: glutamate, lysine, and valine. In contrast to positions 92, 123, and 119, at which basic residues appear to be required for constitutive activation, any replacement of the aspartate at position 115 elevated the basal activity of the receptor (Fig. 5Go, D and E). In fact, even replacement of the aspartate with glutamate constitutively activated the receptor, demonstrating the importance of the size of the residue as well as the charge. Constitutive activation by mutations at this position did not appear to be consistently associated with a reduction in ligand efficacy, since, for example, the EC50s for activation of the D115E and D115K receptors by NDP-{alpha}-MSH were comparable to wild-type values (Table 1Go). While D115E had an affinity for ligand comparable to the wild-type receptor, replacement of the acidic residue with either valine or lysine produced a 2.5- and 100-fold increase in IC50, respectively (Fig. 5FGo and Table 1Go).

Removal of All Three Acidic Residues, E92, D115, and D119, Still Yields a Constitutively Active Receptor
Insertion of a basic residue with a possible positive charge in the second or third membrane-spanning domain could be argued to create an electrostatic interaction between the two membrane-spanning domains that stabilizes the active receptor conformation. To test this hypothesis, we constructed four different receptors with two or three basic residue substitutions. Even when all three conserved acidic residues, E92, D115, and D119, are replaced with lysines, the receptor remains constitutively activated. Thus, once insertion of a single basic residue occurs, removal of the remaining acid residues does not inactivate the receptor. The most potent constitutive activation appeared to occur when both transmembrane domains 2 and 3 were each replaced with a single basic residue. Additionally, insertion of multiple basic residues appeared to remove responsiveness even to the potent ligand NDP-{alpha}-MSH (Fig. 6Go, A and B).



View larger version (13K):
[in this window]
[in a new window]
 
Figure 6. Pharmacology of the Multiple Basic Residue Change Mutants of the mMC1-R

A, {alpha}-MSH stimulation curves for the mouse MC1-R wild-type and multiple basic residue change mutants. All mutant receptors are constitutively active. No EC50 values could be determined. Methods are as in Fig. 2AGo. B, NDP-{alpha}-MSH stimulation curves for the mouse MC1-R wild-type and multiple basic residue change mutants. NDP-{alpha}-MSH could not further activate the mutant receptors. Methods are as in Fig. 2AGo.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The E92K mutation in the Eso-3J allele of the murine MC1-R was originally proposed to constitutively activate the receptor by disrupting an intramolecular electrostatic interaction that constrained the receptor in an inactive conformation (28), in parallel with the constraint demonstrated to exist between K296 and E113 of rhodopsin (29). The data presented here argue strongly against this original model. First, if the glutamic acid residue at position 92 is essential for the formation of an electrostatic interaction that constrains the receptor in an inactive conformation, then any change that removes the acidic residue at this position should cause constitutive activation of the MC1-R. However, only the insertion of a lysine or arginine at this position activated the receptor, while alanine, aspartate, isoleucine, and glutamate did not (Fig. 2Go, A and B). Second, we were unable to find any conserved basic residues in the receptor that could serve as a potential counterion for E92 (Fig. 3Go). Seven mutations in total were made at His183, His258, and Lys276, and none of them caused constitutive activation of the MC1-R.

Remarkably, two additional residues, C125 and D119, were identified with properties very similar to E92. C125R is a mutation found as a dominant allele of the MC1-R in the darkly pigmented Alaska silver fox (42). When this mutation was introduced into the mMC1-R (C123R), the receptor was constitutively activated (Fig. 4Go, A and B). Again, only changes to lysine or arginine resulted in constitutive activation. A change from cysteine to alanine resulted in a receptor that was identical to the wild type, suggesting that the disulfide-bonding capability of the cysteine does not play a role in ligand binding or receptor activation. Similar to the effects seen at E92 and C123, only the introduction of a positive charge at D119 caused constitutive activation of the receptor (Fig. 5Go, A and B).

E92, D115, and D119 are conserved in all five melanocortin receptors and are the only conserved acidic residues existing in the presumed exterior part of the transmembrane domains that would thus be in a position to readily interact as counterions with the essential arginine residue in the H-F-R-W pharmacophore of {alpha}-MSH (9). Independent modeling studies of the MC1-R have suggested this acidic domain as part of the ligand-binding pocket (11, 24). Introduction of nonbasic residues at positions 92 and 115 had no effect on the affinity of ligand binding as assessed in competition binding studies. In contrast, all three D119 variants constructed demonstrated a 10- to 100-fold lower affinity for NDP-{alpha}-MSH or {alpha}-MSH, pointing to this residue as a likely site of electrostatic interaction for arginine 8 of ligand (Fig. 5CGo and Table 1Go).

Constitutively activating mutations in GPCRs have often been found to have increased affinity for, and increased sensitivity to, agonist (16, 27, 31, 37, 43). Surprisingly, the opposite was the case for activating mutations at E92, C123, and D119 of the MC1-R. Insertion of basic residues at these positions uniformly decreased apparent affinity for {alpha}-MSH and NDP-{alpha}-MSH and greatly decreased the efficacy of these agonists (Figs. 2Go, 4Go, and 5Go). Scatchard analysis of one mutation, E92K, demonstrated a 10-fold decrease in affinity of the receptor for NDP-{alpha}-MSH from 0.62 nM to 6.4 nM (Fig. 2DGo) with little change in receptor number. In some cases, agonist efficacy at the constitutively activated MC1 receptors could only be demonstrated with the use of the superpotent agonist NDP-{alpha}-MSH.

Two proline mutations near the extracellular margin of the second membrane-spanning domain have also been found associated with dominant extension locus alleles, L98P in the sombre mouse (5, 28), and L99P in darkly pigmented cattle (17). The L98P receptor showed the same pharmacological properties as the E92K, C123R, and D119K mutant receptors (Figs. 2Go, 4Go, and 5Go). These data suggest that the packing of the second and third transmembrane domains is critical for activation of the MC1-R.

Given the potential proximity of E92, D115, and D119 in space, it is possible that significant repulsion results between TM2 and TM3. Introduction of a basic residue by mutagenesis or by ligand binding could thus reduce this repulsion between the transmembrane domains as part of the conformational change involved in forming R*. Both the E92K/D115K and E92K/C123R double-mutant receptors have basal levels of cAMP-dependent ß-galactosidase activity that are greater than that of either mutation alone, suggesting that addition of basic residues constitutively activates the receptor via independent and additive mechanisms (Fig. 6Go). Furthermore, even replacement of all three conserved acidic residues, E92, D115, and D119, with lysines produces a constitutively active receptor that has now lost any detectable responsiveness, even to the superpotent ligand NDP-{alpha}-MSH (Fig. 6BGo). These data make it difficult to support a model in which any specific interaction between the acidic residues in transmembrane domains 2 and 3 is disrupted as part of the mechanism of constitutive activation. Rather, the mutations may be independently altering the structures of the TM2 and TM3 helices.

In contrast to mutations in the acidic domain, at which basic residues are required for constitutive activation, replacement of the aspartate at D115 with any other residue will cause constitutive activation (Fig. 5Go, D–F). This observation suggests that the D115 may be involved in an electrostatic interaction or hydrogen bond that may serve to constrain the receptor in an inactive conformation. It is likely that D115 is not in a transmembrane milieu, but rather in the first extracellular loop. As such, the constraining interaction may involve residues in this or other extracellular loops that have not yet been studied. Additionally, constitutively activating mutations at this position did not consistently decrease ligand affinity or efficacy, suggesting a different mechanism of action from the L98P, E92K, C123R, and D119K mutations.

According to the ternary allosteric complex model, constitutively activating mutations of the GPCRs should have a reduction in the energy barrier that must be overcome in the R to R* transition (19, 31). Early studies of adrenergic receptors identified several residues in the third intracellular loop, known to be a region involved in G protein coupling (6), that enhanced the basal activity while also demonstrating increasing agonist affinity and efficacy (6, 27, 31). Many subsequent activating mutations characterized in the TSH receptor (43) and LH receptor (37) share this property, and it has been suggested that increased agonist affinity is a hallmark of constitutive activation. However, this did not take into account the possibility of mutations that would be more proximal to ligand binding, decreasing the energy barrier to R* by directly mimicking the conformational effects of ligand binding, and disrupting a component of high-affinity ligand binding in the process. Based on the data presented here, we propose that constitutive activation of the MC1-R by mutations at E92, C123, L98, and D119 is occurring by ligand mimicry. According to this model, the arginine residue at position 8 of {alpha}-MSH, known to be essential for high-affinity ligand binding, normally binds in the pocket formed by these residues, interacting electrostatically with D119. Replacement of E92, C123, or D119 with a basic residue thus mimics the effects of ligand arginine on receptor conformation.

Furthermore, introduction of a basic residue with possible positive charge into the arginine-binding pocket did not appear to release a specific receptor constraint involving molecular interactions between E92, D119, and D115. Substitution of acidic residues with lysines in each transmembrane domain 2 and 3 of the MC1-R appeared to be additive, and even substitution of all three acidic residues produced a constitutively active receptor, suggesting that the relative degree of electrostatic attraction or repulsion of transmembrane domains 2 and 3 is not a critical factor in MC1 receptor activation. Consequently, we propose that one effect of ligand binding, or constitutive activation by ligand mimicry, may be the direct transmission of a conformational change along the TM2 and/or TM3 {alpha}-helical bundles. This could be envisioned as a release from a vertical constraint imposed on the TM {alpha}-helical domains, perhaps by interaction of the charged E92, D119, and D115 residues with the negatively charged lipid head groups at the junctions of the membrane with the aqueous intracellular and extracellular milieu. This information could be transmitted across the membrane in a fashion that ultimately affects the environment of the conserved E/DRY sequence at the beginning of second intracellular loop. Significant evidence exists for rhodopsin (26), as well as the {alpha}1-adrenergic receptor (33, 34), that the protonation state of the glutamic/aspartic acid within the E/DRY sequence is dramatically affected by local changes in the milieu, and the protonation state of this residue in turn appears to have a critical impact on G protein coupling. Mutations in the MC1-R TM2 may indirectly affect protonation state of the aspartic acid by altering the positioning of TM2 residues, such as M71 and D82 of the MC1-R, since comparable residues in the adrenergic receptors (34) and rhodopsin (26) are known to be involved in the formation of a polar pocket that affects the protonation state of the TM3 aspartate. Mutations in TM3 could directly alter the lipophilic environment of the DRY sequence by directly altering the packing of this {alpha}-helix. In conclusion, we provide data here for a corollary of the ternary allosteric model; constitutively activating mutations that are ligand mimetic may increase the fraction of receptors in R* while decreasing agonist affinity and efficacy by virtue of disruption of the ligand-binding site.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Site-Directed Mutagenesis
PCR was used to make single amino acid changes in different regions of the mMC1-R (15). The PBS vector (Strategene, La Jolla, CA) containing the wild-type mMC1-R coding region was amplified using Vent DNA polymerase (New England Biolabs, Beverly, MA) with two adjacent primers designed to hybridize to opposite strands. One primer contained the designed mutation, and the other was complementary to the wild-type receptor. A portion of agarose gel-purified PCR product was phosphorylated and self-ligated. A single colony was selected after transformation. The mutations, as well as the remainder of the mMC1-R, were confirmed by sequencing using an ABI model sequencer (ABI Advanced Biotechnologies, Columbia, MD).

Receptor Expression
The BamHI-SalI fragments from the wild-type mMC1-R and different mMC1-R mutants were cloned into the eukaryotic expression vector pCDNA3 (Invitrogen, San Diego, CA). Human embryonic kidney 293 cells were transfected with 20 µg DNA of each construct using the calcium phosphate method (1). Selection began 72 h posttransfection in DMEM containing 10% newborn calf serum and 1 mg/ml geneticin.

ß-Galactosidase Activity Assay
HEK 293 stable cell populations expressing the wild-type mMC1 receptor or different mutants were transfected with a pCRE/ß-galactosidase (pCRE/ß-gal) construct using the calcium phosphate method (1). Four micrograms of pCRE/ß-gal DNA was used for transfection of a 10 cm dish of cells. At 15–24 h posttransfection, cells were split into 96-well plates with 20,000 to 30,000 cells per well and incubated at 37 C in a 5% CO2 incubator until 48 h posttransfection. Cells were then stimulated with different concentrations of {alpha}-MSH and NDP-{alpha}-MSH diluted in stimulation medium (DMEM containing 0.1 mg/ml BSA and 0.1 mM isobutylmethylxanthine) for 6 h at 37 C in a 5% CO2 incubator. The cells were also stimulated by 10 µM forskolin to normalize for transfection efficiency. After stimulation, cells were lysed in 50 µl lysis buffer (250 mM Tris-HCl, pH 8.0, 0.1% Triton X-100), frozen and thawed, and then assayed for ß-galactosidase activity as described (2). ß-Galactosidase activity was normalized to protein concentration and displayed as a fold of forskolin (10 µM)-stimulated level to normalize for efficiency of transfection of the receptor construct. Previous work has demonstrated that ß-galactosidase activity is proportional to intracellular cAMP over a wide range of physiological concentrations (2). Data represent means and SDs from triplicate data points, and curves were fitted by nonlinear regression using Prism software (GraphPAD, San Diego, CA).

Ligand Binding
Competition binding experiments were performed on stable cell populations containing the wild or mutant receptors. Cells were plated at 5 x 106 cells per well in 24-well plates the day before the binding experiment was performed. The cells were then incubated for 45 min at room temperature in binding medium (1 mg/ml BSA in Ca2+/Mg2+ PBS) containing 30,000-100,000 cpm of [125I]NDP-{alpha}-MSH per well. Series concentrations of unlabeled NDP-{alpha}-MSH or {alpha}-MSH were used to compete with the labeled NDP-{alpha}-MSH. Controls for nonspecific binding contained 1 or 10 µM unlabeled NDP-{alpha}-MSH or {alpha}-MSH. After 45 min of incubation, the medium was aspirated, and the cells were washed once with 1 ml of BSA/PBS (1 mg/ml BSA in Ca2+/Mg2+ PBS) per well. Later, 0.5 ml versine (GIBCO, Grand Island, NY) was used to transfer cells to test tubes for counting radioactivity. Data represent means and SDs from duplicate data points, and curves were fitted by nonlinear regression using Prism software (GraphPAD).

Saturation binding experiments were performed on stable cell lines containing the wild-type or E92K mutant receptors. Increased concentrations of [125I]NDP-{alpha}-MSH (100,000 cpm to 1,000,000 cpm) in binding medium (1 mg/ml BSA in Ca2+/Mg2+ PBS) were added to cells in the absence and presence of 10-4 M unlabeled {alpha}-MSH. The cells were incubated at room temperature for 45 min. After the incubation, cells were washed twice with 1 ml of BSA/PBS (1 mg/ml BSA in Ca2+/Mg2+ PBS) per well. Later, 0.5 ml of versine was used twice to transfer cells to test tubes for counting radioactivity. Data represent means and SDs from duplicate data points, and curves were fitted by linear regression using Prism software (GraphPAD).


    FOOTNOTES
 
Address requests for reprints to: Roger D. Cone, Vollum Institute, 3181 Southwest Sam Jackson Park Road, Portland, Oregon 97201.

This work was supported by NIH Grant AR-42415 (R.D.C.) and the Norwegian Fur Breeder’s Association (D.I.V.).

Received for publication September 2, 1997. Revision received November 28, 1997. Accepted for publication January 13, 1998.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Chen C, Okayama H 1987 High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7:2745–2752[Medline]
  2. Chen W, Shields TS, Stork PJ, Cone RD 1995 A colorimetric assay for measuring activation of Gs- and Gq-coupled signaling pathways. Anal Biochem 226:349–354[CrossRef][Medline]
  3. Cohen GB, Oprian DD, Robinson PR 1992 Mechanism of activation and inactivation of opsin: role of Glu113 and Lys296. Biochemistry 31:12592–12601[Medline]
  4. Cohen GB, Yang T, Robinson PR, Oprian DD 1993 Constitutive activation of opsin: influence of charge at position 134 and size at position 296. Biochemistry 32:6111–6115[Medline]
  5. Cone RD, Lu D, Chen W, Koppula S, Vage DI, Klungland H, Boston B, Orth DN, Pouton C, Kesterson RA 1996 The melanocortin receptors: agonists, antagonists, and the hormonal control of pigmentation. Recent Prog Horm Res 51:287–318[Medline]
  6. Cotecchia S, Exum S, Caron MG, Lefkowitz RJ 1990 Regions of the alpha 1-adrenergic receptor involved in coupling to phosphatidylinositol hydrolysis and enhanced sensitivity of biological function. Proc Natl Acad Sci USA 87:2896–2900[Abstract]
  7. DeLean A, Stadel JM, Lefkowitz RJ 1980 A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase-coupled ß-adrenergic receptor. J Biol Chem 255:7108–7117[Abstract/Free Full Text]
  8. Dryja TP, Berson EL, Rao VR, Oprian DD 1993 Heterozygous missense mutation in the rhodopsin gene as a cause of congenital stationary night blindness. Nat Genet 4:280–284[Medline]
  9. Eberle AN 1988 The Melanotropins: Chemistry, Physiology and Mechanisms of Action. Karger, Basel, Switzerland
  10. Gether U, Lin S, Kobilka BK 1995 Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes. J Biol Chem 270:28268–28275[Abstract/Free Full Text]
  11. Haskell-Luevano C, Sawyer TK, Trumpp-Kallmeyer S, Bikker J, Humblet C, Gantz I, Hruby VJ 1996 Three-dimensional molecular models of the hMC1R melanocortin receptor: complexes with melanotropin peptide agonists. Drug Design Discovery 14:197–211[Medline]
  12. Hearing VJ 1987 Mammalian monophenol monooxygenase (tyrosinase): purification, properties and reactions catalyzed. In: Kaufman S (ed) Methods in Enzymology-Metabolism of Aromatic Amino Acids and Amines. Academic Press, New York, pp 154–165
  13. Hearing VJ, Jimenez M 1987 Mammalian tyrosinase: the critical regulatory control point in melanocyte pigmentation. Int J Biochem 19:1141–1147[CrossRef][Medline]
  14. Hearing VJ, Tsukamoto K 1991 Enzymatic control of pigmentation in mammals. FASEB J 5:2902–9209[Abstract/Free Full Text]
  15. Imai Y, Matsushima Y, Sugimura T, Terada M 1991 Asimple and rapid method for generating a deletion by PCR. Nucleic Acids Res 19:2785[Medline]
  16. Kjelsberg MA, Cotecchia S, Ostrowski J, Caron MG, Lefkowitz RJ 1992 Constitutive activation of the alpha 1B-adrenergic receptor by all amino acid substitutions at a single site. Evidence for a region which constrains receptor activation. J Biol Chem 267:1430–1433[Abstract/Free Full Text]
  17. Klungland H, Vage DI, Gomez-Raya L, Adalsteinsson S, Lien S 1995 The role of melanocyte-stimulating hormone (MSH) receptor in bovine coat color determination. Mammalian Genome 6:636–639[Medline]
  18. Laue L, Chan W-Y, Hsueh AJW, Kudo M, Hsu SY, Wu S-M, Blomberg L, Cutler J, GB 1995 Genetic heterogeneity of constitutively activating mutations of the human lutenizing hormone receptor in familial male-limited precocious puberty. Proc Natl Acad Sci USA 92:1906–1910[Abstract]
  19. Lefkowitz RJ, Cotecchia S, Samama P, Costa T 1993 Constitutive activity of receptors coupled to guanine nucleotide regulatory proteins. Trends Pharmacol Sci 14:303–307[CrossRef][Medline]
  20. Nathans J 1990 Determinants of visual pigment absorbance: identification of the retinylidene Schiff’s base counterion in bovine rhodopsin. Biochemistry 29:9746–9752[Medline]
  21. Parma J, Duprez L, Van Sande J, Cochaux P, Gervy C, Mockel J, Dumont J, Vassart G 1993 Somatic mutations in the thyrotropin receptor gene cause hyperfunctioning thyroid adenomas. Nature 365:649–651[CrossRef][Medline]
  22. Pollak MR, Brown EM, Estep HL, McLaine PN, Kifor O, Park J, Hebert SC, Seidman CE, Seidman JG 1994 Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation. Nat Genet 8:303–307[Medline]
  23. Porcellini A, Ciullo I, Pannain S, Fenzi G, Avvedimento E 1995 Somatic mutations in the VI transmembrane segment of the thyrotropin receptor constitutively activate cAMP signalling in thyroid hyperfunctioning adenomas. Oncogene 11:1089–1093[Medline]
  24. Prusis P, Frändberg P-A, Muceniece R, Kalvinsh I, Wikberg JES 1995 A three dimensional model for the interaction of MSH with the melanocortin-1 receptor. Biochem Biophys Res Commun 210:205–210[CrossRef][Medline]
  25. Rao VR, Cohen GB, Oprian DD 1994 Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness. Nature 367:639–642[CrossRef][Medline]
  26. Rao VR, Oprian DD 1996 Activating mutations of rhodopsin and other G protein-coupled receptors. Annu Rev Biophys Biomol Struct 25:287–314[CrossRef][Medline]
  27. Ren Q, Kurose H, Lefkowitz RJ, Cotecchia S 1993 Constitutively active mutants of the alpha 2-adrenergic receptor. J Biol Chem 268:16483–16487[Abstract/Free Full Text]
  28. Robbins LS, Nadeau JH, Johnson KR, Kelly MA, Roselli-Rehfuss L, Baack E, Mountjoy KG, Cone RD 1993 Pigmentation phenotypes of variant extension locus alleles result from point mutations that alter MSH receptor function. Cell 72:827–834[Medline]
  29. Robinson PR, Cohen GB, Zhukovsky EA, Oprian DD 1992 Constitutively active mutants of rhodopsin. Neuron 9:719–725[Medline]
  30. Sakmar TP, Franke RR, Khorana HG 1989 Glutamic acid-113 serves as the retinylidene Schiff base counterion in bovine rhodopsin. Proc Natl Acad Sci USA 86:8309–8313[Abstract]
  31. Samama P, Cotecchia S, Costa T, Lefkowitz RJ 1993 A mutation-induced activated state of the beta 2-adrenergic receptor. Extending the ternary complex model. J Biol Chem 268:4625–4636[Abstract/Free Full Text]
  32. Sawyer TK, Sanfilippo PJ, Hruby VJ, Engel MH, Heward CB, Burnett JB, Hadley ME 1980 4-Norleucine, 7-D-phenylalanine-alpha-melanocyte-stimulating hormone: a highly potent alpha-melanotropin with ultralong biological activity. Proc Natl Acad Sci USA 77:5754–5758[Abstract]
  33. Scheer A, Fanelli F, Costa T, De Benedetti PG, Cotecchia S 1997 The activqtion process of the alpha1B-adrenergic receptor: potential role of protonation and hydrophobicity of a highly conserved aspartate. Proc Natl Acad Sci USA 94:808–813[Abstract/Free Full Text]
  34. Scheer A, Fanelli F, Costa T, De Benedetti PG, Cotecchia S 1996 Constitutively active mutants of the alpha 1B-adrenergic receptor: role of highly conserved polar amino acids in receptor activation. EMBO J 15:3566–3578[Abstract]
  35. Schipani E, Kruse K, Juppner H 1995 A constitutively active mutant PTH-PTHrP receptor in Jansen-type metaphyseal chondrodysplasia. Science 268:98–100[Medline]
  36. Searle AG 1968 Comparative Genetics of Coat Colors in Mammals. Logos Press Ltd, London
  37. Shenker A, Laue L, Kosugi S, Merendino Jr JJ, Minegishi T, Cutler Jr GB 1993 A constitutively activating mutation of the luteinizing hormone receptor in familial male precocious puberty. Nature 365:652–654[CrossRef][Medline]
  38. Takeuchi S, Suzuki H, Yabuuchi M, Takahashi S 1996 A possible involvement of melanocortin 1-receptor in regulating feather color pigmentation in the chicken. Biochim Biophys Acta 1308:164–168[Medline]
  39. Tominaga Y, Takagi H 1996 Molecular genetics of hyperparathyroid disease. Curr Opin Nephrol Hyper 5:336–341[Medline]
  40. Tonacchera M, Van Sande J, Cetani F, Swillens S, Schvartz C, Winiszewski P, Portmann L, Dumont JE, Vassart G, Parma J 1996a Functional characteristics of three new germline mutations of the thyrotropin receptor gene causing autosomal dominant toxic thyroid hyperplasia. J Clin Endocrinol Metab 81:547–554
  41. Tonacchera M, Van Sande J, Parma J, Duprez L, Cetani F, Costagliola S, Dumont JE, Vassart G 1996b TSH receptor and disease. Clin Endocrinol (Oxf) 44:621–633
  42. Vage DI, Lu D, Klungland H, Lien S, Adalsteinsson S, Cone RD 1997 A non-epistatic interaction of agouti and extension in the fox, Vulpes vulpes. Nat Genet 15:311–315[Medline]
  43. Van Sande J, Parma J, Tonacchera M, Swillens S, Dumont J, Vassart G 1995 Somatic and germline mutations of the TSH receptor gene in thyroid diseases. J Clin Endocrinol Metab 80:2577–2585[Medline]
  44. Vassart G, Desarnaud F, Duprez L, Eggerickx D, Labbe O, Libert F, Mollereau C, Parma J, Paschke R, Tonacchera M, Vanderhaeghen P, Van Sande J, Dumont J, Parmentier M 1995 The G protein-coupled receptor family and one of its members, the TSH receptor. Ann NY Acad Sci 766:23–30[Medline]
  45. Yano K, Saji M, Hidaka A, Moriya N, Okuno A, Kohn LD, Cutler J, GB 1995 A new constitutively activating point mutation in the luteinizing hormone/choriogonadotropin receptor gene in cases of male-limited precocious puberty. J Clin Endocrinol Metab 80:1162–1168[Abstract]