Regulation of G protein-coupled cAMP receptor activation by a hydrophobic residue in transmembrane helix 3

cAR1, a G protein-coupled cAMP receptor, is essential for multicellular development of Dictyostelium. We previously identified a cAR1-Ile(104) mutant that appeared to be constitutively activated based on its constitutive phosphorylation, elevated affinity for cAMP, and dominant-negative effects on development as well as specific cAR1 pathways that are subject to adaptation. To investigate how Ile(104) might regulate cAR1 activation, we assessed the consequences of substituting it with all other amino acids. Constitutive phosphorylation of these Ile(104) mutants varied broadly, suggesting that they are activated to varying extents, and was correlated with polarity of the substituting amino acid residue. Remarkably, all Ile(104) substitutions, except for the most conservative, dramatically elevated the receptor's cAMP affinity. However, only a third of the mutants (those with the most polar substitutions) blocked development. These findings are consistent with a model in which polar Ile(104) substitutions perturb the equilibrium between inactive and active cAR1 conformations in favour of the latter. Based on homology with rhodopsin, Ile(104) is likely buried within inactive cAR1 and exposed to the cytoplasm upon activation. We propose that the hydrophobic effect normally promotes burial of Ile(104) and hence cAR1 inactivation, while polar substitution of Ile(104) mitigates this effect, resulting in 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.     

The extracellular N-terminal domain and transmembrane domains 1 and 2 mediate oligomerization of a yeast G protein-coupled receptor

G protein-coupled receptors (GPCRs) can form homodimers/oligomers and/or heterodimers/oligomers. The mechanisms used to form specific GPCR oligomers are poorly understood because the domains that mediate such interactions and the step(s) in the secretory pathway where oligomerization occurs have not been well characterized. Here we have used subcellular fractionation and fluorescence resonance energy transfer (FRET) experiments to show that oligomerization of a GPCR (alpha-factor receptor; STE2 gene product) of the yeast Saccharomyces cerevisiae occurs in the endoplasmic reticulum. To identify domains of this receptor that mediate oligomerization, we used FRET and endocytosis assays of oligomerization in vivo to analyze receptor deletion mutants. A mutant lacking the N-terminal extracellular domain and transmembrane (TM) domain 1 was expressed at the cell surface but did not self-associate. In contrast, a receptor fragment containing only the N-terminal extracellular domain and TM1 could self-associate and heterodimerize with wild type receptors. Analysis of other mutants suggested that oligomerization is facilitated by the N-terminal extracellular domain and TM2. Therefore, the N-terminal extracellular domain, TM1, and TM2 appear to stabilize alpha-factor receptor oligomers. These domains may form an interface in contact or domain-swapped oligomers. Similar domains may mediate dimerization of certain mammalian GPCRs.     

A microdomain formed by the extracellular ends of the transmembrane domains promotes activation of the G protein-coupled alpha-factor receptor

The alpha-factor receptor (Ste2p) that promotes mating in Saccharomyces cerevisiae is similar to other G protein-coupled receptors (GPCRs) in that it contains seven transmembrane domains. Previous studies suggested that the extracellular ends of the transmembrane domains are important for Ste2p function, so a systematic scanning mutagenesis was carried out in which 46 residues near the ends of transmembrane domains 1, 2, 3, 4, and 7 were replaced with cysteine. These mutants complement mutations constructed previously near the ends of transmembrane domains 5 and 6 to analyze all the extracellular ends. Eight new mutants created in this study were partially defective in signaling (V45C, N46C, T50C, A52C, L102C, N105C, L277C, and A281C). Treatment with 2-([biotinoyl] amino) ethyl methanethiosulfonate, a thiol-specific reagent that reacts with accessible cysteine residues but not membrane-embedded cysteines, identified a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. An unusual prolonged zone of intermediate reactivity near the extracellular end of transmembrane domain 2 suggests that this region may adopt a special structure. Interestingly, residues implicated in ligand binding were mainly accessible, whereas residues involved in the subsequent step of promoting receptor activation were mainly inaccessible. These results define a receptor microdomain that provides an important framework for interpreting the mechanisms by which functionally important residues contribute to ligand binding and activation of Ste2p and other GPCRs.     

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.     

Aromatic residues at the extracellular ends of transmembrane domains 5 and 6 promote ligand activation of the G protein-coupled alpha-factor receptor

The alpha-factor receptor (STE2) stimulates a G protein signaling pathway that promotes mating of the yeast Saccharomyces cerevisiae. Previous random mutagenesis studies implicated residues in the regions near the extracellular ends of the transmembrane domains in ligand activation. In this study, systematic Cys scanning mutagenesis across the ends of transmembrane domains 5 and 6 identified two residues, Phe(204) and Tyr(266), that were important for receptor signaling. These residues play a specific role in responding to alpha-factor since the F204C and Y266C substituted receptors responded to an alternative agonist (novobiocin). To better define the structure of this region, the Cys-substituted mutant receptors were assayed for reactivity with a thiol-specific probe that does not react with membrane-imbedded residues. A drop in reactivity coincided with residues likely to be buried in the membrane. Interestingly, both Phe(204) and Tyr(266) are located very near the interface region. However, these assays predict that Phe(204) is accessible at the surface of the receptor, consistent with the strong defect in binding alpha-factor caused by mutating this residue. In contrast, Tyr(266) was not accessible. This correlates with the ability of Y266C mutant receptors to bind alpha-factor and suggests that this residue is involved in the subsequent triggering of receptor activation. These results highlight the role of aromatic residues near the ends of the transmembrane segments in the alpha-factor receptor, and suggest that similar aromatic residues may play an important role in other G protein-coupled receptors.     

Opioid receptor random mutagenesis reveals a mechanism for G protein-coupled receptor activation

The high resolution structure of rhodopsin has greatly enhanced current understanding of G protein-coupled receptor (GPCR) structure in the off-state, but the activation process remains to be clarified. We investigated molecular mechanisms of delta-opioid receptor activation without a preconceived structural hypothesis. Using random mutagenesis of the entire receptor, we identified 30 activating point mutations. Three-dimensional modeling revealed an activation path originating from the third extracellular loop and propagating through tightly packed helices III, VI and VII down to a VI-VII cytoplasmic switch. N- and C-terminal determinants also influence receptor activity. Findings for this therapeutically important receptor may apply to other GPCRs that respond to diffusible ligands.     

Mechanism of activation of a G protein-coupled receptor, the human cholecystokinin-2 receptor

G protein-coupled receptors (GPCRs) represent a major focus in functional genomics programs and drug development research, but their important potential as drug targets contrasts with the still limited data available concerning their activation mechanism. Here, we investigated the activation mechanism of the cholecystokinin-2 receptor (CCK2R). The three-dimensional structure of inactive CCK2R was homology-modeled on the basis of crystal coordinates of inactive rhodopsin. Starting from the inactive CCK2R modeled structure, active CCK2R (namely cholecystokinin-occupied CCK2R) was modeled by means of steered molecular dynamics in a lipid bilayer and by using available data from other GPCRs, including rhodopsin. By comparing the modeled structures of the inactive and active CCK2R, we identified changes in the relative position of helices and networks of interacting residues, which were expected to stabilize either the active or inactive states of CCK2R. Using targeted molecular dynamics simulations capable of converting CCK2R from the inactive to the active state, we delineated structural changes at the atomic level. The activation mechanism involved significant movements of helices VI and V, a slight movement of helices IV and VII, and changes in the position of critical residues within or near the binding site. The mutation of key amino acids yielded inactive or constitutively active CCK2R mutants, supporting this proposed mechanism. Such progress in the refinement of the CCK2R binding site structure and in knowledge of CCK2R activation mechanisms will enable target-based optimization of nonpeptide ligands.     

A conserved Asn in transmembrane helix 7 is an on/off switch in the activation of the thyrotropin receptor

The thyrotropin (TSH) receptor is an interesting model to study G protein-coupled receptor activation as many point mutations can significantly increase its basal activity. Here, we identified a molecular interaction between Asp(633) in transmembrane helix 6 (TM6) and Asn(674) in TM7 of the TSHr that is crucial to maintain the inactive state through conformational constraint of the Asn. We show that these residues are perfectly conserved in the glycohormone receptor family, except in one case, where they are exchanged, suggesting a direct interaction. Molecular modeling of the TSHr, based on the high resolution structure of rhodopsin, strongly favors this hypothesis. Our approach combining site-directed mutagenesis with molecular modeling shows that mutations disrupting this interaction, like the D633A mutation in TM6, lead to high constitutive activation. The strongly activating N674D (TM7) mutation, which in our modeling breaks the TM6-TM7 link, is reverted to wild type-like behavior by an additional D633N mutation (TM6), which would restore this link. Moreover, we show that the Asn of TM7 (conserved in most G protein-coupled receptors) is mandatory for ligand-induced cAMP accumulation, suggesting an active role of this residue in activation. In the TSHr, the conformation of this Asn residue of TM7 would be constrained, in the inactive state, by its Asp partner in TM6.     

The chain length dependence of helix formation of the second transmembrane domain of a G protein-coupled receptor of Saccharomyces cerevisiae

The chain length dependence of helix formation of transmembrane peptides in lipids was investigated using fragments corresponding to the second transmembrane domain of the alpha-factor receptor from Saccharomyces cerevisiae. Seven peptides with chain lengths of 10 (M2-10; FKYLLSNYSS), 14 (M2-14), 18 (M2-18), 22 (M2-22), 26 (M2-26), 30 (M2-30) and 35 (M2-35; RSRKTPIFIINQVSLFLIILHSALYFKYLLSNYSS) residues, respectively, were synthesized. CD spectra revealed that M2-10 was disordered, and all of the other peptides assumed partially alpha-helical secondary structures in 99% trifluoroethanol (TFE)/H(2)O. In 50% TFE/H(2)O, M2-30 assumed a beta-like structure. The other six peptides exhibited the same CD patterns as those found in 99% TFE/H(2)O. In 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1 ratio) vesicles, M2-22, M2-26, and M2-35 formed alpha-helical structures, whereas the other peptides formed beta-like structures. Fourier transform infrared spectroscopy in 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dimyristoyl-sn-glycero-3-phospho-rac-(1-glycerol) (4:1) multilayers showed that M2-10, M2-14, M2-18, and M2-30 assumed beta-structures in this environment. Another homologous 30-residue peptide (M2-30B), missing residues SNYSS from the N terminus and extending to RSRKT on the C terminus, was helical in lipid bilayers, suggesting that residues at the termini of transmembrane domains influence their biophysical properties. Attenuated total reflection Fourier transform infrared spectroscopy revealed that M2-22, M2-26, M2-30B, and M2-35 were alpha-helical and oriented at angles of 12 degrees, 13 degrees, 36 degrees, and 34 degrees, respectively, with respect to the multilayer normal. This study showed that chain length must be taken into consideration when using peptides representing single transmembrane domains as surrogates for regions of an intact receptor. Furthermore, this work indicates that the tilt angle and conformation of transmembrane portions of G protein-coupled receptors may be estimated by detailed spectroscopic measurements of single transmembrane peptides.     

A conformational trigger for activation of a G protein by a G protein-coupled receptor

G protein-coupled receptors (GPCRs) are a family of seven transmembrane helical proteins that initiate a cellular response to an environmental signal. Once activated by an extracellular signal, GPCRs trigger the intracellular signal transduction cascade by activating a heterotrimeric G protein. The interaction between the G protein and the receptor, which triggers the signal transduction, is the focus of intense interest. Three-dimensional structures of the ground state of only one GPCR, rhodopsin, are currently available, but since the G protein cannot bind to this structure, these structures did not lead to an understanding of the activation process. The recent publication of an excited state structure for the same GPCR (and comparison to the ground state structures), in conjunction with other recent biochemical data, provides new insight into G protein activation. We find that the structure data and the biochemical data, for the first time, point to a specific mode of interaction between the G protein and the receptor. Furthermore, we find that transducin (G(t)) must alter its conformation to bind to the activated receptor; the "lock and key" fit heretofore expected is likely not the correct model. We suggest that a conformational distortion, driven by the energy of binding, is induced in G(t) when it binds to the activated receptor. The conformational change in turn enables the exchange of GTP for GDP and the dissociation of the subunits. This is an example of "induced fit" originally proposed by Koshland to describe enzyme-substrate interactions.     

A proposed structure for transmembrane segment 7 of G protein-coupled receptors incorporating an asn-Pro/Asp-Pro motif.

Transmembrane segment (TMS) 7 has been shown to play an important role in the signal transduction function of G-protein-coupled receptors (GPCRs). Although transmembrane segments are most likely to adopt a helical structure, results from a variety of experimental studies involving TMS 7 are inconsistent with it being an ideal alpha-helix. Using results from a search of the structure database and extensive simulated annealing Monte Carlo runs with the new Conformational Memories method, we have identified the conserved (N/D)PxxY region of TMS 7 as the major determinant for deviation of TMS 7 from ideal helicity. The perturbation consists of an Asx turn and a flexible "hinge" region. The Conformational Memories procedure yielded a model structure of TMS 7 which, unlike an ideal alpha-helix, is capable of accommodating all of the experimentally derived geometrical criteria for the interactions of TMS 7 in the transmembrane bundle of GPCRs. In the context of the entire structure of a transmembrane bundle model for the 5HT2a receptor, the specific perturbation of TMS 7 by the NP sequence suggests a structural hypothesis for the pattern of amino acid conservation observed in TMS 1, 2, and 7 of GPCRs. The structure resulting from the incorporation of the (N/D)P motif satisfies fully the H-bonding capabilities of the 100% conserved polar residues in these TMSs, in agreement with results from mutagenesis experiments. The flexibility introduced by the specific structural perturbation produced by the (NP/DP) motif in TMS 7 is proposed to have a significant role in receptor activation.     

Molecular basis for activation of G protein-coupled receptor kinases

G protein‐coupled receptor (GPCR) kinases (GRKs) selectively recognize and are allosterically regulated by activated GPCRs, but the molecular basis for this interaction is not understood. Herein, we report crystal structures of GRK6 in which regions known to be critical for receptor phosphorylation have coalesced to stabilize the kinase domain in a closed state and to form a likely receptor docking site. The crux of this docking site is an extended N‐terminal helix that bridges the large and small lobes of the kinase domain and lies adjacent to a basic surface of the protein proposed to bind anionic phospholipids. Mutation of exposed, hydrophobic residues in the N‐terminal helix selectively inhibits receptor, but not peptide phosphorylation, suggesting that these residues interact directly with GPCRs. Our structural and biochemical results thus provide an explanation for how receptor recognition, phospholipid binding, and kinase activation are intimately coupled in GRKs.     

The role of G beta gamma and domain interfaces in the activation of G protein-coupled receptor kinase 2

In response to extracellular signals, G protein-coupled receptors (GPCRs) catalyze guanine nucleotide exchange on Galpha subunits, enabling both activated Galpha and Gbetagamma subunits to target downstream effector enzymes. One target of Gbetagamma is G protein-coupled receptor kinase 2 (GRK2), an enzyme that initiates homologous desensitization by phosphorylating activated GPCRs. GRK2 consists of three distinct domains: an RGS homology (RH) domain, a protein kinase domain, and a pleckstrin homology (PH) domain, through which it binds Gbetagamma. The crystal structure of the GRK2-Gbetagamma complex revealed that the domains of GRK2 are intimately associated and left open the possibility for allosteric regulation by Gbetagamma. In this paper, we report the 4.5 A structure of GRK2, which shows that the binding of Gbetagamma does not induce large domain rearrangements in GRK2, although small rotations of the RH and PH domains relative to the kinase domain are evident. Mutation of residues within the larger domain interfaces of GRK2 generally leads to diminished expression and activity, suggesting that these interfaces are important for stability and remain intact upon activation of GRK2. Geranylgeranylated Gbetagamma, but not a soluble mutant of Gbetagamma, protects GRK2 from clostripain digestion at a site within its kinase domain that is 80 A away from the Gbetagamma binding site. Equilibrium ultracentrifugation experiments indicate that neither abnormally large detergent micelles nor protein oligomerization can account for the observed protection. The Gbetagamma-mediated binding of GRK2 to CHAPS micelles or lipid bilayers therefore appears to rigidify the kinase domain, perhaps by encouraging stable contacts between the RH and kinase domains.     

Residues in the first extracellular loop of a G protein-coupled receptor play a role in signal transduction

The Saccharomyces cerevisiae pheromone, alpha-factor (WHWLQLKPGQPMY), and Ste2p, its G protein-coupled receptor, were used as a model system to study ligand-receptor interaction. Cys-scanning mutagenesis on each residue of EL1, the first extracellular loop of Ste2p, was used to generate a library of 36 mutants with a single Cys residue substitution. Mutation of most residues of EL1 had only negligible effects on ligand affinity and biological activity of the mutant receptors. However, five mutants were identified that were either partially (L102C and T114C) or severely (N105C, S108C, and Y111C) compromised in signaling but retained binding affinities similar to those of wild-type receptor. Three-dimensional modeling, secondary structure predictions, and subsequent circular dichroism studies on a synthetic peptide with amino acid sequence corresponding to EL1 suggested the presence of a helix corresponding to EL1 residues 106 to 114 followed by two short beta-strands (residues 126 to 135). The distinctive periodicity of the five residues with a signal-deficient phenotype combined with biophysical studies suggested a functional involvement in receptor activation of a face on a 3(10) helix in this region of EL1. These studies indicate that EL1 plays an important role in the conformational switch that activates the Ste2p receptor to initiate the mating pheromone signal transduction pathway.     

Deorphanization of Dresden G protein-coupled receptor for an odorant receptor

Dresden G protein-coupled receptor (D-GPCR) is one of orphan G protein-coupled receptors (GPCR). Here we report the identification of the ligands and the characterization of D-GPCR. We investigated over 5000 compounds to evoke the response mediated by D-GPCR and identified 3-methyl-valeric acid and 4-methyl-valeric acid as agonists using a cAMP assay. It is of interest that they dramatically enhanced the intracellular cAMP accumulation and the CRE-luciferase activity in CHO-K1 cells and HEK293 cells expressing the chimeric protein of D-GPCR with a rhodopsin-tag at its N-terminus. Our results established new characteristics of D-GPCR as an olfactory receptor. First, agonists of D-GPCR belong to odorants. Second, D-GPCR mRNA is expressed in the olfactory bulb. In addition, D-GPCR was reported to have similar sequences and its genome locus nearby other olfactory receptors. These results suggest D-GPCR is an olfactory receptor.     

Synthesis and biophysical analysis of transmembrane domains of a Saccharomyces cerevisiae G protein-coupled receptor

The Ste2p receptor for alpha-factor, a tridecapeptide mating pheromone of the yeast Saccharomyces cerevisiae, belongs to the G protein-coupled family of receptors. In this paper we report on the synthesis of peptides corresponding to five of the seven transmembrane domains (M1-M5) and two homologues of the sixth transmembrane domain corresponding to the wild-type sequence and a mutant sequence found in a constitutively active receptor. The secondary structures of all new transmembrane peptides and previously synthesized peptides corresponding to domains 6 and 7 were assessed using a detailed CD analysis in trifluoroethanol, trifluoroethanol-water mixtures, sodium dodecyl sulfate micelles, and dimyristoyl phosphatidyl choline bilayers. Tryptophan fluorescence quenching experiments were used to assess the penetration of the membrane peptides into lipid bilayers. All peptides were predominantly (40-80%) helical in trifluoroethanol and most trifluoroethanol-water mixtures. In contrast, two of the peptides M3-35 (KKKNIIQVLLVASIETSLVFQIKVIFTGDNFKKKG) and M6-31 (KQFDSFHILLINleSAQSLLVPSIIFILAYSLK) formed stable beta-sheet structures in both sodium dodecyl sulfate micelles and DMPC bilayers. Polyacrylamide gel electrophoresis showed that these two peptides formed high molecular aggregates in the presence of SDS whereas all other peptides moved as monomeric species. The peptide (KKKFDSFHILLIMSAQSLLVLSIIFILAYSLKKKS) corresponding to the sequence in the constitutive mutant was predominantly helical under a variety of conditions, whereas the homologous wild-type sequence (KKKFDSFHILLIMSAQSLLVPSIIFILAYSLKKKS) retained a tendency to form beta-structures. These results demonstrate a connection between a conformational shift in secondary structure, as detected by biophysical techniques, and receptor function. The aggregation of particular transmembrane domains may also reflect a tendency for intermolecular interactions that occur in the membrane environment facilitating formation of receptor dimers or multimers.     

Structural requirements of the extracellular to transmembrane domain junction for erythropoietin receptor function

The erythropoietin receptor (EpoR) is crucial for erythrocyte formation. The x-ray crystal structures of the EpoR extracellular domain lack the juxtamembrane (JM) region and the junction to the transmembrane (TM) domain. Yet the JM-TM regions are important for transmitting the conformational change imposed on the receptor dimer by Epo binding. Cysteine-scanning mutagenesis of the JM-TM regions identified three novel constitutively active mutants, demonstrating close disulfide-bonded juxtapositioning of these residues in the JM (L223C) and N-terminal TM domain (L226C, I227C). Chemical cross-linking defined the interface of the active helical TM dimer and revealed that the JM-TM segment encompassing Leu(226)-Leu(230) is non-helical. Molecular dynamics and NMR studies indicated that the TM-JM junction forms an N-terminal helix cap. This structure is important for EpoR function because replacement of this motif by consecutive leucines rendered the receptor constitutively active.     

Regulation of EGF-induced ERK/MAPK activation and EGFR internalization by G protein-coupled receptor kinase 2

G protein-coupled receptor kinases （GRKs） mediate agonist-induced phosphorylation and desensitization of various G protein-coupled receptors （GPCRs）. We investigate the role of GRK2 on epidermal growth factor （EGF） receptor signaling, including EGF-induced extracellular signal-regulated kinase and mitogen-activated protein kinase （ERK/MAPK） activation and EGFR internalization. Immunoprecipitation and immunofluorescence experiments show that EGF stimulates GRK2 binding to EGFR complex and GRK2 translocating from cytoplasm to the plasma membrane in human embryonic kidney 293 cells. Western blotting assay shows that EGF-induced ERK/MAPK phosphorylation increases 1.9-fold, 1.1-fold and 1.5fold （P〈0.05） at time point 30, 60 and 120 min, respectively when the cells were transfected with GRK2,suggesting the regulatory role of GRK2 on EGF-induced ERK/MAPK activation. Flow cytometry experiments show that GRK2 overexpression has no effect on EGF-induced EGFR internalization, however, it increases agonist-induced G protein-coupled δ5 opioid receptor internalization by approximately 40% （P〈0.01）. Overall,these data suggest that GRK2 has a regulatory role in EGF-induced ERK/MAPK activation, and that the mechanisms underlying the modulatory role of GRK2 in EGFR and GPCR signaling pathways are somewhat different at least in receptor internalization.     

Allosteric activation of the extracellular Ca2+-sensing receptor by L-amino acids enhances ERK1/2 phosphorylation

The calcium-sensing receptor (CaR) mediates feedback control of Ca2+o (extracellular Ca2+) concentration. Although the mechanisms are not fully understood, the CaR couples to several important intracellular signalling enzymes, including PI-PLC (phosphoinositide-specific phospholipase C), leading to Ca2+i (intracellular Ca2+) mobilization, and ERK1/2 (extracellular-signal-regulated kinase 1/2). In addition to Ca2+o, the CaR is activated allosterically by several subclasses of L-amino acids, including the aromatics L-phenylalanine and L-tryptophan. These amino acids enhance the Ca2+o-sensitivity of Ca2+i mobilization in CaR-expressing HEK-293 (human embryonic kidney) cells and normal human parathyroid cells. Furthermore, on a background of a physiological fasting serum L-amino acid mixture, they induce a small, but physiologically significant, enhancement of Ca2+o-dependent suppression of PTH (parathyroid hormone) secretion. The impact of amino acids on CaR-stimulated ERK1/2, however, has not been determined. In the present study, we examined the effects of L-amino acids on Ca2+o-stimulated ERK1/2 phosphorylation as determined by Western blotting and a newly developed quantitative assay (SureFire). L-Amino acids induced a small, but significant, enhancement of Ca2+o-stimulated ERK1/2. In CaR-expressing HEK-293 cells, 10 mM L-phenylalanine lowered the EC50 for Ca2+o from approx. 2.3 to 2.0 mM in the Western blot assay and from 3.4 to 2.9 mM in the SureFire assay. The effect was stereoselective (L>D), and another aromatic amino acid, L-tryptophan, was also effective. The effects of amino acids were investigated further in HEK-293 cells that expressed the CaR mutant S169T. L-Phenylalanine normalized the EC50 for Ca2+o-stimulated Ca2+i mobilization from approx. 12 mM to 5.0 mM and ERK1/2 phosphorylation from approx. 4.6 mM to 2.6 mM. Taken together, the data indicate that L-phenylalanine and other amino acids enhance the Ca2+o-sensitivity of CaR-stimulated ERK1/2 phosphorylation; however, the effect is comparatively small and operates in the form of a fine-tuning mechanism.