Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.     

The formation of a salt bridge between helices 3 and 6 is responsible for the constitutive activity and lack of hormone responsiveness of the naturally occurring L457R mutation of the human lutropin receptor

The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.     

Constitutive activation of G protein-coupled receptors as a result of selective substitution of a conserved leucine residue in transmembrane helix III

Whereas numerous mutations of the human lutropin receptor (hLHR) and human TSH receptor (hTSHR) have been shown to cause constitutive activation of these receptors, it has been suggested that either the hFSHR as a whole, or the i3/TM VI region of the hFSHR, is less susceptible to mutation-induced constitutive activation. However, as shown herein, substitution of a highly conserved leucine residue in transmembrane III (TM III) of the hFSHR (Leu 111.18) with arginine causes a 5-fold increase in basal cAMP in transfected cells, consistent with a strong constitutive activation of the hFSHR. Interestingly, this mutant is unresponsive to further hormonal stimulation. Substitutions of hFSHR(L460) with lysine, alanine, or aspartate show that only arginine causes constitutive activation. However, all result in decreased FSH responsiveness, suggesting a role for L460 in FSH-stimulated cAMP production by the hFSHR. Because Leu 111.18 is highly conserved in rhodopsin-like G protein-coupled receptors (GPCRs), we tested the effects of substitution of the comparable leucine in the human beta2-adrenergic receptor (hbeta2-AR). Substitution of L124 in the hbeta2-AR with arginine, lysine, or alanine resulted in constitutive activation as evidenced by increased basal levels of cAMP that could be attenuated by an inverse agonist. In all cases, isoproterenol-stimulated cAMP was unaffected. Taken altogether, our data support a model whereby Leu 111.18 may play a general role in GPCRs by stabilizing them in an inactive state. Constitutive activation may arise by both a disruption of Leu 111.18 as well as the introduction of a specific residue that serves to stabilize the active state of the receptor.     

A constitutively active mutant of the human lutropin receptor (hLHR-L457R) escapes lysosomal targeting and degradation

Using biochemical and imaging approaches, we examined the postendocytotic fate of the complex formed by human choriogonadotropin (hCG) and a constitutively active mutant of the human lutropin receptor (hLHR-L457R) found in a boy with precocious puberty and Leydig cell hyperplasia. After internalization, some of the complex formed by the hLHR-wild type (hLHR-wt) and hCG recycles to the cell surface, and some is found in lysosomes where the hormone is degraded. In contrast, the complex formed by the hLHR-L457R and hCG is not routed to the lysosomes, most of it is recycled to the cell surface and hormone degradation is barely detectable. For both, hLHR-wt and -L457R, there is an hCG-induced loss of cell surface receptors that accompanies internalization but this loss cannot be prevented by leupeptin. The removal of recycling motifs of the hLHR by truncation of the C-terminal tail at residue 682 greatly enhances the lysosomal accumulation of the hormone-receptor complexes formed by the hLHR-wt or the L457R mutant, the degradation of the internalized hormone, and the loss of cell surface receptors. The degradation of the hormone internalized by these mutants as well as the loss of cell surface receptors is largely prevented by leupeptin. These results highlight a previously unrecognized complexity in the postendocytotic trafficking of the hLHR and document a clear difference between the properties of the constitutively active mutant and the agonist-activated hLHR-wt. This lack of lysosomal degradation of the L457R mutant could contribute to its constitutive activity by prolonging the duration of signaling.     

A cell surface inactive mutant of the human lutropin receptor (hLHR) attenuates signaling of wild-type or constitutively active receptors via heterodimerization

The D405N and Y546F mutations of the human lutropin receptor (hLHR) have previously been shown to partially attenuate hCG-stimulated cAMP synthesis despite normal cell surface expression and hCG binding affinity (Min, L. and Ascoli, M. Mol. Endocrinol. 14:1797–1810, 2000). We now show that these mutations each stabilize a resting state of the hLHR. A combined mutant D405N,Y546F is similarly expressed at the cell surface and exhibits normal ligand-binding, but is profoundly signaling impaired. Introduction of hLHR(wt) into cells stably expressing the signaling inactive D405N,Y546F resulted in the attenuation of hCG-stimulated cAMP production by hLHR(wt) even if excess Gs is co-expressed. Similarly, co-expression of D405N,Y546F with hLHR constitutively active mutants (CAMs) attenuated their constitutive activity. Quantitative bioluminescence resonance energy transfer (BRET) analyses demonstrated that D405N,Y546F formed heterodimers with both wt and CAM hLHR. In contrast hLHR(D405N,Y546F) did not heterodimerize with the melanocortin 3 receptor (MC3R) and agonist-stimulated cAMP production through the MC3R was not attenuated when these two receptors were co-expressed. Taken altogether, our data demonstrate that a signaling inactive hLHR mutant (that is trafficked normally to the plasma membrane) attenuates the signaling of the cell surface localized wt or the constitutively active hLHR due to receptor heterodimerization. Our studies, therefore, suggest a novel ramification of GPCR signaling resulting from receptor dimerization.     

Effects of truncations of the cytoplasmic tail of the luteinizing hormone/chorionic gonadotropin receptor on receptor-mediated hormone internalization.

The LH/CG receptor is a member of the family of G protein-coupled receptors and consists of a large N-terminal extracellular domain (which is responsible for binding hormone) attached to a region that spans the plasma membrane seven times, ending with an intracellularly located C-terminus. Binding of LH or human CG (hCG) to the LH/CG receptor causes a stimulation of adenylyl cyclase, presumably via activation of Gs. The binding of hormone also leads to its subsequent internalization by receptor-mediated endocytosis. In order to investigate the role of the cytoplasmic tail of this receptor in these events, we prepared a series of mutants in which progressively larger portions of the cytoplasmic tail were deleted. Deletion of 58 amino acids from the C-terminus, in which only 11 cytoplasmic residues remain, resulted in a receptor that was not expressed on the plasma membrane. Receptors rat LHR (rLHR)-t653 and rLHR-t631, in which 21 or 43 amino acids were removed, respectively, were properly expressed. These results suggest that a region(s) between residues 616 and 631 of the rLH/CG receptor are required for proper insertion and/or targeting of the receptor into the plasma membrane. Cells expressing rLHR-t653 or rLHR-t631 bound hCG with the same high affinity as cells expressing the full-length receptor, and basal levels of cAMP were the same among the cells. However, cells expressing the truncated receptors responded to hCG with approximately 2-fold greater levels of maximal cAMP accumulation than cells expressing the full-length receptor. Deletion of up to 43 amino acids from the C-terminus of the rLH/CG receptor had no deleterious effect on hCG internalization. In fact, mutants lacking 21 and 43 amino acids exhibited progressively faster rates of hCG internalization as compared to the full-length receptor. Once internalized, hCG was also degraded at a faster rate in cells expressing the truncated LH/CG receptors. Since hCG-stimulated cAMP stimulation and hCG internalization are retained by rLHR-t631, it can be concluded that the residues, not necessarily the same, required for these functions reside within the 26 amino acids of the cytoplasmic tail closest to the seventh transmembrane helix and/or residues within the intracellular loops. Our data show, however, that both hCG-stimulated cAMP production and hCG internalization are enhanced by the removal of the distal portion of the cytoplasmic tail.     

Molecular mechanism underlying partial and full agonism mediated by the human cholecystokinin-1 receptor

The cholecystokinin-1 receptor (CCK1R) is a G protein-coupled receptor (GPCR) that regulates important physiological functions. As for other GPCRs, the molecular basis of full and partial agonism is still far from clearly understood. In the present report, using both laboratory experiments and molecular modeling approaches, we have investigated the partial agonism mechanism of JMV 180, on the human CCK1R. We first showed that efficacy of the CCK1R to activate phospholipase C is dependent on the correct orientation of the C-terminal end of peptidic ligands toward residue Phe(330) of helix VI. We have previously reported that a single mutation of Met(121) (helix III) markedly reduced the receptor-mediated inositol phosphate production upon stimulation by CCK. Computational simulations predicted that residue 121 affected orientation of the C-terminal end of CCK, thus suggesting that the molecular complex with a reduced inositol phosphate production observed with the mutated CCK1R resembles that resulting from binding of JMV 180 to the WT-CCK1R. Pharmacological, biochemical, and functional characterizations of the two receptor.ligand complexes with decreased abilities to signal were carried out in different cell types. We found that they presented the same features, such as total dependence of inositol phosphate production to Galpha(q) expression, single affinity of binding sites, insensitivity of binding to non-hydrolyzable GTP, absence of GTPgamma[S(35)] binding following agonist stimulation, similarity of dose-response curves for amylase secretion, and incapacity to induce acute pancreatitis in pancreatic acini. We concluded that helices VI and III of the CCK1R are functionally linked through the CCK1R agonist binding site and that positioning of the C-terminal ends of peptidic agonists toward Phe(330) of helix VI is responsible for extent of phospholipase C activation through Galpha(q) coupling. Given the potential therapeutic interest of partial agonists such as JMV 180, our structural data will serve for target structure-based design of new CCK1R ligands.     

Intrinsic differences in the response of the human lutropin receptor versus the human follitropin receptor to activating mutations

In contrast to the human lutropin receptor (hLHR), very few naturally occurring activating mutations of the structurally related human follitropin receptor (hFSHR) have been identified. The present study was undertaken to determine if one aspect underlying this discrepancy might be a general resistance of the hFSHR to mutation-induced constitutive activity. Five different mutations were introduced into both the hLHR and hFSHR (four based on activating mutations of the hLHR gene, one based on an activating mutation of the hFSHR gene). Our results demonstrate that hFSHR constitutively activating mutants (CAMs) were not as active as hLHR CAMs containing the comparable mutation. Furthermore, although all hFSHR CAMs exhibited strong promiscuous activation by high concentrations of the other glycoprotein hormone receptors, hLHR CAMs showed little or no promiscuous activation. Our in vitro findings are consistent with in vivo observations of known pathophysiological conditions associated with hLHR CAMs, but not hFSHR CAMs, and with promiscuous activation of hFSHR CAMs, but not hLHR CAMs. Computational experiments suggest that the mechanisms through which homologous mutations increase the basal activity of the hLHR and the hFSHR are similar. This is particularly true for the strongest CAMs like L460(3.43)R. Disparate properties of the hLHR versus hFSHR CAMs may, therefore, be due to differences in shape and electrostatics features of the solvent-exposed cytosolic receptor domains involved in the receptor-G protein interface rather than to differences in the nature of local perturbation at the mutation site or in the way local perturbation is transferred to the putative G protein binding domains.     

Mutations of the second extracellular loop of the human lutropin receptor emphasize the importance of receptor activation and de-emphasize the importance of receptor phosphorylation in agonist-induced internalization

Alanine scanning mutagenesis of the second extracellular loop of the human lutropin receptor (hLHR) showed that mutation of most of the residues present in this region either enhance or impair the internalization of agonist. A more complete analysis of four mutants, two that enhanced internalization (F515A and T521A) and two that impaired internalization (S512A and V519A), showed that the two mutants that impaired internalization also show a decrease in the sensitivity for agonist-induced cAMP accumulation, whereas the two mutants that enhanced internalization show an increase in the sensitivity for agonist-induced cAMP accumulation. None of these mutants had an effect on the agonist-induced phosphorylation of the hLHR, however. We conclude that, in contrast to the prevailing view of the relative importance of receptor phosphorylation in the internalization of G protein-coupled receptors, the phosphorylation of the hLHR is less important than the agonist-induced activation of the hLHR in the process of internalization.     

Aliphatic amino acids in helix VI of the AT(1) receptor play a relevant role in agonist binding and activity

Angiotensin II (AII) AT(1) receptor mutants with replacements of aliphatic amino acids in the distal region of helix VI and the adjoining region of the third extracellular loop (EC-3) were expressed in Chinese hamster ovary (CHO) cells to determine their role in ligand binding and activation. The triple mutant [L262D, L265D, L268D]AT(1) (L3D) showed a marked reduction in affinity for AII and for non-peptide (losartan) and peptide ([Sar(1)Leu(8) ]AII) antagonists; in functional assays using inositol phosphate (IP) accumulation, the relative potency and the maximum effect of AII were reduced in L3D. Replacement of Leu(268) (in EC-3) and Leu(262) (in the transmembrane domain) by aspartyl residues did not cause significant changes in the receptor's affinity for the ligands and in IP production. In contrast, the point mutation L265D, at helix VI, markedly decreased affinity and ability to stimulate phosphatidylinositol turnover. Molecular modeling of the AT(1) receptor based on a recent crystal structure of rhodopsin, suggests that the side chain of Leu(265) but not that of Leu(262) is facing a cleft between helices V and VI and interacts with the lipid bilayer, thus helping to stabilize the receptor structure near the Lys(199) residue of helix V in the agonist binding site which is necessary for full activity.     

Characterization of Three Vasopressin Receptor 2 Variants: An Apparent Polymorphism (V266A) and Two Loss-of-Function Mutations (R181C and M311V)

Arginine vasopressin (AVP) is released from the posterior pituitary and controls water homeostasis. AVP binding to vasopressin V2 receptors (V2Rs) located on kidney collecting duct epithelial cells triggers activation of Gs proteins, leading to increased cAMP levels, trafficking of aquaporin-2 water channels, and consequent increased water permeability and antidiuresis. Typically, loss-of-function V2R mutations cause nephrogenic diabetes insipidus (NDI), whereas gain-of-function mutations cause nephrogenic syndrome of inappropriate antidiuresis (NSIAD). Here we provide further characterization of two mutant V2Rs, R181C and M311V, reported to cause complete and partial NDI respectively, together with a V266A variant, in a patient diagnosed with NSIAD. Our data in HEK293FT cells revealed that for cAMP accumulation, AVP was about 500- or 30-fold less potent at the R181C and M311V mutants than at the wild-type receptor respectively (and about 4000- and 60-fold in COS7 cells respectively). However, in contrast to wild type V2R, the R181C mutant failed to increase inositol phosphate production, while with the M311V mutant, AVP exhibited only partial agonism in addition to a 37-fold potency decrease. Similar responses were detected in a BRET assay for β-arrestin recruitment, with the R181C receptor unresponsive to AVP, and partial agonism with a 23-fold decrease in potency observed with M311V in both HEK293FT and COS7 cells. Notably, the V266A V2R appeared functionally identical to the wild-type receptor in all assays tested, including cAMP and inositol phosphate accumulation, β-arrestin interaction, and in a BRET assay of receptor ubiquitination. Each receptor was expressed at comparable levels. Hence, the M311V V2R retains greater activity than the R181C mutant, consistent with the milder phenotype of NDI associated with this mutant. Notably, the R181C mutant appears to be a Gs protein-biased receptor incapable of signaling to inositol phosphate or recruiting β-arrestin. The etiology of NSIAD in the patient with V266A V2R remains unknown.     

Arrestin-3 is essential for the activation of Fyn by the luteinizing hormone receptor (LHR) in MA-10 cells

Recent studies showed that Fyn is a mediator of the LHR-induced activation of the ERK1/2 cascade in MA-10 cells. Since the LHR is a G protein-coupled receptor and the Src family of kinases can be activated by some Galpha subunits and by the non-visual arrestins we investigated the role of these signaling molecules in the LHR-provoked activation of Fyn. Small interfering RNAs (siRNAs) that target two Galpha subunits that participate in LHR signaling (Galpha(s) and Galpha(11)) and one that targets arrestin-3 were co-transfected with the hLHR in MA-10 cells. We then determined the effects of these siRNAs on the LHR-provoked activation of Fyn, the phosphorylation of FAK (a prominent Fyn substrate) and the release of EGF-like growth factors (a Fyn-mediated process). Expression of the siRNA against Galpha(s) decreased the level of Galpha(s) and LHR-stimulated cAMP production by approximately 50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Likewise, expression of the siRNA against Galpha(11) decreased the level of Galpha(11) and LHR-stimulated inositol phosphate production by approximately 50% but did not affect LHR-stimulated Fyn activation or FAK phosphorylation. Expression of the siRNA against arrestin-3 decreased the level of arrestin-3 and the rate of internalization of hCG by approximately 50% and it also inhibited the LHR-provoked stimulation of Fyn, the phosphorylation of FAK and the release of EGF-like growth factors. These results show that, in MA-10 cells, the hLHR activates Fyn through an arrestin-3-dependent pathway and that this pathway is a mediator of the hLHR-provoked release of EGF-like growth factors.     

Association of human follitropin (FSH) receptor with splicing variant of human lutropin/choriogonadotropin receptor negatively controls the expression of human FSH receptor

A splice variant of human lutropin (LH)/choriogonadotropin (CG)-receptor [hLHR(exon 9)] that lacks exon 9 was previously cloned in the corpus luteum of a woman with a normal menstrual cycle. Supported by a detergent-soluble binding assay and a receptor biotinylation experiment, the receptor binding assay shows hLHR(exon 9) is neither expressed at the cell surface nor has the capability of binding to hCG. In addition, hLHR(exon 9) was confirmed in the endoplasmic reticulum (ER) by endoglycosidase H treatment. A coimmunoprecipitation experiment clearly showed that hLHR(exon 9) and constitutively inactivate mutant-LHRs, which stay in the ER, form an association with the human follitropin (FSH)-receptor (hFSHR). This suggests that in the presence of mutant-LHR, hFSHR, which is trapped in the ER and associated with hLHR(exon 9), is unable to come up to the plasma membrane. This phenomenon is specific among gonadotropin receptors because human TSH receptor failed to be coimmunoprecipitated. Furthermore, this receptor complex attenuated the hFSHR receptor protein level within the cells, which impaired cAMP production. To elucidate the mechanism underlying the decrease in hFSHR protein by this receptor complex, we performed a Percoll fractionation experiment, which indicated that the receptor complex drove hFSHR to the lysosome instead of the plasma membrane. These results reveal a novel mechanism of FSHR expression regulation.     

Activation of the AT1 angiotensin receptor is dependent on adjacent apolar residues in the carboxyl terminus of the third cytoplasmic loop

The C-terminal region of the third intracellular loop of the AT(1) angiotensin receptor (AT(1)-R) is an important determinant of G protein coupling. The roles of individual residues in agonist-induced activation of G(q/11)-dependent phosphoinositide hydrolysis were determined by mutational analysis of the amino acids in this region. Functional studies on mutant receptors transiently expressed in COS-7 cells showed that alanine substitutions of the amino acids in positions 232-240 of the third loop had no major effect on signal generation. However, deletion mutations that removed Ile(238) or affected its position relative to transmembrane helix VI significantly impaired angiotensin II-induced inositol phosphate responses. Substitution of Ile(238) with an acidic residue abolished the ability of the receptor to mediate inositol phosphate production, whereas its replacement with basic or polar residues reduced the amplitude of inositol phosphate responses. Substitutions of Phe(239) with polar residues had relatively minor effects on inositol phosphate signal generation, but its replacement by aspartic acid reduced, and by positively charged residues (Lys, Arg) significantly increased, angiotensin II-induced inositol phosphate responses. The internalization kinetics of the Ile(238) and Phe(239) mutant receptors were impaired in parallel with the reduction in their signaling responses. These findings have identified Ile(238) and Phe(239) as the critical residues in the C-terminal region of the third intracellular loop of the AT(1)-R for receptor activation. They also suggest that an apolar amino acid corresponding to Ile(238) of the AT(1)-R is a general requirement for activation of other G protein-coupled receptors by their agonist ligands.     

Differential conformational requirements for activation of G proteins and the regulatory proteins arrestin and G protein-coupled receptor kinase in the G protein-coupled receptor for parathyroid hormone (PTH)/PTH-related protein

After stimulation with agonist, G protein-coupled receptors (GPCRs) activate G proteins and become phosphorylated by G protein-coupled receptor kinases (GRKs), and most of them translocate cytosolic arrestin proteins to the cytoplasmic membrane. Agonist-activated GPCRs are specifically phosphorylated by GRKs and are targeted for endocytosis by arrestin proteins, suggesting a connection between GPCR conformational changes and interaction with GRKs and arrestins. Previously, we showed that by substitution of histidine for residues at the cytoplasmic side of helix 3 (H3) and helix 6 (H6) of the parathyroid hormone (PTH) receptor (PTHR), a zinc metal ion-binding site is engineered that prevents PTH-stimulated G(s) activation (Sheikh, S. P., Vilardaga, J.-P., Baranski, T. J., Lichtarge, O., Iiri, T., Meng, E. C., Nissenson, R. A., and Bourne, H. R. (1999) J. Biol. Chem. 274, 17033-17041). These data suggest that relative movements between H3 and H6 are critical for G(s) activation. Does this molecular event play a similar role in activation of GRK and arrestin and in PTHR-mediated G(q) activation? To answer this question, we utilized the two previously described mutant forms of PTHR, H401 and H402, which contain a naturally present histidine residue at position 301 in H3 and a second substituted histidine residue at positions 401 and 402 in H6, respectively. Both mutant receptors showed inhibition of PTH-stimulated inositol phosphate and cAMP generation in the presence of increasing concentrations of Zn(II). However, the mutants showed no Zn(II)-dependent impairment of phosphorylation by GRK-2. Likewise, the mutants were indistinguishable from wild-type PTHR in the ability to translocate beta-arrestins/green fluorescent protein to the cell membrane and were also not affected by sensitivity to Zn(II). These results suggest that agonist-mediated phosphorylation and internalization of PTHR require conformational switches of the receptor distinct from the cAMP and inositol phosphate signaling state. Furthermore, PTHR sequestration does not appear to require G protein activation.     

Functional role of transmembrane helix 7 in the activation of the heptahelical lutropin receptor

A member of the G protein-coupled receptor superfamily, the LH receptor (LHR), and the two other glycoprotein hormone receptors are distinguished from the other members by the presence of a relatively large N-terminal extracellular domain that is responsible for high-affinity ligand binding. Transmembrane helix (TMH) 7 of LHR is amphipathic, with an extended face containing only hydrophobic side chains and another containing both hydrophobic and polar side chains with potential hydrogen bond donor and acceptor functions. Since several reports have shown the importance of this helix in ligand-mediated signaling, we have used Ala scanning mutagenesis to study eight amino acid residues of rat LHR that are invariant in the three glycoprotein hormone receptors, Leu586, Val587, Asn593, Ser594, Cys595, Asn597, Phe604, and Thr605. The wild type (WT) and mutant cDNAs were transiently transfected into COS-7 cells for characterization by human CG (hCG) binding and cAMP production. No differences were detected in dissociation constants (K(d)S) or basal cAMP production relative to WT LHR, but three categories of LHR mutants were distinguished from WT LHR based upon their expression levels and responsiveness to hCG: 1) comparable or higher expression but reduced ligand responsiveness (N593A and C595A), 2) reduced expression and ligand responsiveness (N597A and T605A), and 3) comparable expression and responsiveness (L586A, V587A, S594A, and F604A). Three other mutants, C595M, F604Y, and T605Y, were comparable to WT LHR in ligand responsiveness. To provide more information on Asn593 and Asn597, a total of 12 replacements were investigated. Of considerable interest and potential significance was the finding that many of the replacements in LHR resulted in either loss of function (N593A, Q, S; N597R) or gain of function (N593R and N597Q), this being the first evidence of a position in LHR that, depending upon the nature of the amino acid residue, can result in constitutive activation and/or diminished responsiveness to ligand. The results of molecular modeling and energy minimization of TMHs 6 and 7, based on a postulated interaction between Asp556 (TMH 6) and Asn593/Asn597 (TMH 7), indicated that, while there is not a correlation between function and predicted energies of WT LHR and the mutants, reorientation of one or both helices is responsible for the functional changes observed. Possible interactions of TMHs 3 and 4 and of 5 and 6 were suggested by molecular modeling. Ten mutants were prepared of two amino acid residues that are invariant in the glycoprotein hormone receptors and have side chain hydrogen bond donor and acceptor function, Glu429 in TMH 3 and Asn513 in TMH 5. Expression levels and hCG-mediated signaling were reduced in most of the LHR mutants, but none of these exhibited constitutive receptor activation. We conclude that Glu429 is not critical for receptor function, while Asn513 appears to be particularly important in receptor folding and/or trafficking. The results reported herein indicate an important role for TMH 7, and particularly Asn593 and Asn597, in the process of receptor activation. Moreover, these two asparagines, although in close proximity to each other in TMH 7, are quite distinct in function as evidenced by certain replacements that can lead to loss of function in one and gain of function in the other.     

Rescue of misrouted GnRHR mutants reveals its constitutive activity

G protein-coupled receptors (GPCR) play central roles in almost all physiological functions, and mutations in GPCR are responsible for over 30 hereditary diseases associated with loss or gain of receptor function. Gain of function mutants are frequently described as having constitutive activity (CA), that is, they activate effectors in the absence of agonist occupancy. Although many GPCR have mutants with CA, the GnRH receptor (GnRHR) was not, until 2010, associated with any CA mutants. The explanation for the failure to observe CA appears to be that the quality control system of the cell recognizes CA mutants of GnRHR as misfolded and retains them in the endoplasmic reticulum. In the present study, we identified several human (h)GnRHR mutants with substitutions in transmembrane helix 6 (F(272)K, F(272)Q, Y(284)F, C(279)A, and C(279)S) that demonstrate varying levels of CA after being rescued by pharmacoperones from different chemical classes and/or deletion of residue K(191), a modification that increases trafficking to the plasma membrane. The movement of the mutants from the endoplasmic reticulum (unrescued) to the plasma membrane (after rescue) is supported by confocal microscopy. Judging from the receptor-stimulated inositol phosphate production, mutants F(272)K and F(272)Q, after rescue, display the largest level of CA, an amount that is comparable with agonist-stimulated activation. Because mutations in other GPCR are, like the hGnRHR, scrutinized by the quality control system, this general approach may reveal CA in receptor mutants from other systems. A computer model of the hGnRHR and these mutants was used to evaluate the conformation associated with CA.     

A transfected m1 muscarinic acetylcholine receptor stimulates adenylate cyclase via phosphatidylinositol hydrolysis

The m1 muscarinic acetylcholine receptor gene was transfected into and stably expressed in A9 L cells. The muscarinic receptor agonist, carbachol, stimulated inositol phosphate generation, arachidonic acid release, and cAMP accumulation in these cells. Carbachol stimulated arachidonic acid and inositol phosphate release with similar potencies, while cAMP generation required a higher concentration. Studies were performed to determine if the carbachol-stimulated cAMP accumulation was due to direct coupling of the m1 muscarinic receptor to adenylate cyclase via a GTP binding protein or mediated by other second messengers. Carbachol failed to stimulate adenylate cyclase activity in A9 L cell membranes, whereas prostaglandin E2 did, suggesting indirect stimulation. The phorbol ester, phorbol 12-myristate 13-acetate (PMA), stimulated arachidonic acid release yet inhibited cAMP accumulation in response to carbachol. PMA also inhibited inositol phosphate release in response to carbachol, suggesting that activation of phospholipase C might be involved in cAMP accumulation. PMA did not inhibit prostaglandin E2-, cholera toxin-, or forskolin-stimulated cAMP accumulation. The phospholipase A2 inhibitor eicosatetraenoic acid and the cyclooxygenase inhibitors indomethacin and naproxen had no effect on carbachol-stimulated cAMP accumulation. Carbachol-stimulated cAMP accumulation was inhibited with TMB-8, an inhibitor of intracellular calcium release, and W7, a calmodulin antagonist. These observations suggest that carbachol-stimulated cAMP accumulation does not occur through direct m1 muscarinic receptor coupling or through the release of arachidonic acid and its metabolites, but is mediated through the activation of phospholipase C. The generation of cytosolic calcium via inositol 1,4,5-trisphosphate and subsequent activation of calmodulin by m1 muscarinic receptor stimulation of phospholipase C appears to generate the accumulation of cAMP.     

Phe(303) in TMVI of the alpha(1B)-adrenergic receptor is a key residue coupling TM helical movements to G-protein activation.

We showed previously that Phe(303) in transmembrane segment (TM) VI of the alpha(1B)-adrenergic receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M. (2000) EMBO J. 19, 4265-4271]. Here, we show that saturation mutagenesis of Phe(303) results in a series of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed increased basal activity but an attenuated maximal response to (-)-epinephrine-stimulation. F303L, on the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A > WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants result in TM helical movements that differ in accordance with their degree of constitutive activity. These findings, therefore, confirm and extend our previous data implicating Phe(303) as a key residue coupling TM helical movements to G-protein-activation.