G protein-coupled receptors. II. Mechanism of agonist activation.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
U Gether
Publications and source records attributed to U Gether.
Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The Beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.
The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gs alpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gs alpha (Gs alphaL) binds GDP with lower affinity than the short splice variant (Gs alphaS), but the impact of this difference on the interaction of Gs alpha with the beta 2AR is not known. We studied the beta2 AR/Gs alpha interaction using receptor/G-protein fusion proteins (beta2 AR Gs alphaS and beta2 AR Gs alphaL) expressed in Sf9 cells. Fusion of the beta2 AR to Gs alpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2 AR or Gs alpha. The beta2 AR in beta2 AR Gs alphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2 AR in beta2 AR Gs alphaS. The apparent constitutive activity of the beta2 AR in beta2 AR Gs alphaL may be due to the lower GDP affinity of Gs alphaL compared with Gs alphaS, i.e. Gs alphaL is more often nucleotide-free than Gs alphaS and, therefore, more frequently available to stabilize the beta2 AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.
Adrenergic beta-Agonists↗
Different effects of Gsalpha splice variants on beta2-adrenoreceptor-mediated signaling. The beta2-adrenoreceptor coupled to the long splice variant of Gsalpha has properties of a constitutively active receptor.
The beta2-adrenoreceptor (beta2AR) couples to the G-protein Gs to mediate adenylyl cyclase activation. The splice variants of Gsalpha differ by a 15-amino acid insert between the Ras-like domain and the alpha-helical domain. The long splice variant of Gsalpha (GsalphaL) binds GDP with lower affinity than the short splice variant (GsalphaS), but the impact of this difference on the interaction of Gsalpha with the beta2AR is not known. We studied the beta2AR/Gsalpha interaction using receptor/G-protein fusion proteins (beta2ARGsalphaS and beta2ARGsalphaL) expressed in Sf9 cells. Fusion of the beta2AR to Gsalpha promotes efficient coupling as shown by high-affinity agonist binding and GTPase and adenylyl cyclase activation and ensures fixed stoichiometry between receptor and G-protein. Importantly, fusion does not change the fundamental properties of the beta2AR or Gsalpha. The beta2AR in beta2ARGsalphaL showed hallmarks of constitutive activity (increased potency and intrinsic activity of partial agonists, increased efficacy of inverse agonists, and increased basal GTPase activity) compared with the beta2AR in beta2ARGsalphaS. The apparent constitutive activity of the beta2AR in beta2ARGsalphaL may be due to the lower GDP affinity of GsalphaL compared with GsalphaS, i.e. GsalphaL is more often nucleotide-free than GsalphaS and, therefore, more frequently available to stabilize the beta2AR in the active (R*) state. This study demonstrates that subtle structural differences between closely related G-protein alpha-subunits can have important consequences for the functional properties of a G-protein-coupled receptor.
Adenylyl Cyclases↗
Examination of ligand-induced conformational changes in the beta2 adrenergic receptor.
The environmentally sensitive and cysteine reactive fluorescent probe, IANBD, was used to monitor ligand-induced structural changes in the beta2 adrenergic receptor (beta2AR) by fluorescent spectroscopy. We found that agonists caused a dose-dependent and reversible decrease in fluorescence from the purified IANBD-labeled beta2AR. This suggested that agonists promote a conformational change in the receptor that leads to an increase in the polarity of the environment around one or more IANBD labeled cysteines. The wildtype receptor contains eight free cysteines and mutagenesis and peptide mapping experiments have indicated that several of these sites are accessible for chemical derivatization. Thus, to identify the cysteine(s) involved in the agonist-induced change in fluorescence and thereby map agonist-induced conformational changes in the beta2AR, we generated a series of mutant receptors having limited numbers of cysteines available for fluorescent labeling. Fluorescence spectroscopy analysis of the purified and site-selectively IANBD-labeled mutants showed that IANBD labeled 125Cys and 285Cys are responsible for the observed changes in fluorescence consistent with movements of TM III and VI in response to agonist binding.
Adrenergic beta-Agonists↗
Examination of ligand-induced conformational changes in the beta 2-adrenergic receptor by fluorescence spectroscopy.
Explore the source record for details and available documents.
Adenylyl Cyclases↗
Agonists induce conformational changes in transmembrane domains III and VI of the beta2 adrenoceptor.
Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the beta2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant beta2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.
Adrenergic beta-Agonists↗
Structural instability of a constitutively active G protein-coupled receptor. Agonist-independent activation due to conformational flexibility.
Mutations in several domains can lead to agonist-independent, constitutive activation of G protein-coupled receptors. However, the nature of the structural and molecular changes that constitutively turn on a G protein-coupled receptor remains unknown. Here we show evidence that a constitutively activated mutant of the beta2 adrenergic receptor (CAM) is characterized by structural instability and an exaggerated conformational response to ligand binding. The structural instability of CAM could be demonstrated by a 4-fold increase in the rate of denaturation of purified receptor at 37 degrees C as compared with the wild type receptor. Spectroscopic analysis of purified CAM labeled with the conformationally sensitive and cysteine-reactive fluorophore, N,N'dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)ethylenediamine, further indicated that both agonist and antagonist elicit more profound structural changes in CAM than in the wild type protein. We propose that the mutation that confers constitutive activity to the beta2 adrenergic receptor removes some stabilizing conformational constraints, allowing CAM to more readily undergo transitions between the inactive and the active states and making the receptor more susceptible to denaturation.
Adrenergic beta-Agonists↗
Ligand stabilization of the beta 2 adrenergic receptor: effect of DTT on receptor conformation monitored by circular dichroism and fluorescence spectroscopy.
Treatment of the beta 2 adrenergic receptor with the reducing agent dithiothreitol (DTT) is known to abolish ligand binding to the receptor. Interestingly, the loss of binding can be prevented by preoccupation of the receptor with ligand. It is unclear, however, whether the ligand blocks access of DTT to the receptor, or the ligand stabilizes the receptor structure. In the present study, we have utilized circular dichroism (CD) and intrinsic tryptophan fluorescence to directly probe structural changes in the beta 2 adrenergic receptor in response to DTT treatment. Analysis of CD spectra of purified beta 2 receptor in the detergent micelle indicated that the receptor has an alpha-helix content of 60%, which is substantially more than what would be attributed to the seven transmembrane domains. The alpha-helix content was unchanged in the presence of DTT, suggesting that DTT treatment does not alter the secondary structure of the receptor. In contrast, the tryptophan fluorescence spectra demonstrated that DTT induces a reversible conformational change of the beta 2 receptor. Thus, DTT caused a red-shift in the maximum emission wavelength of the intrinsic tryptophan fluorescence. The change in emission spectrum correlated with a loss in the ability of the receptor to bind antagonist. Both changes in receptor binding and fluorescence emission were reversible, as removal of DTT allowed the receptor to restore 70% of ligand binding and return to the initial emission spectrum. Furthermore, we found adrenergic antagonists were able to slow the rate of the conformational change induced by DTT but not the rate of disulfide reduction, suggesting that the antagonists stabilize the structure of the reduced receptor.
Amino Acid Sequence↗
Fluorescent labeling of purified beta 2 adrenergic receptor. Evidence for ligand-specific conformational changes.
The purpose of the present study was to develop an approach to directly monitor structural changes in a G protein-coupled receptor in response to drug binding. Purified human beta 2 adrenergic receptor was covalently labeled with the cysteine-reactive, fluorescent probe N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)ethylenediamine (IANBD). IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment. We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta 2 receptor. The change in fluorescence could be plotted against the concentration of isoproterenol as a simple hyperbolic binding isotherm demonstrating interaction with a single binding site in the receptor. The ability of several adrenergic antagonists to reverse the response confirmed that this binding site is identical to the well described binding site in the beta 2 receptor. Comparison of the response to isoproterenol with a series of adrenergic agonists, having different biological efficacies, revealed a linear correlation between biological efficacy and the change in fluorescence. This suggests that the agonist-mediated decrease in fluorescence from IANBD-labeled beta 2 receptor is due to the same conformational change as involved in receptor activation and G protein coupling. In contrast to agonists, negative antagonists induced a small but significant increase in base-line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure. In conclusion, our data provide the first direct evidence for ligand-specific conformational changes occurring in a G protein-coupled receptor. Furthermore, the data demonstrate the potential of fluorescence spectroscopy as a tool for further delineating the molecular mechanisms of drug action at G protein-coupled receptors.
Adrenergic beta-Agonists↗
Tachykinin non-peptide antagonists: binding domain and molecular mode of action.
Explore the source record for details and available documents.
Animals↗
Mutations along transmembrane segment II of the NK-1 receptor affect substance P competition with non-peptide antagonists but not substance P binding.
Mutational analysis of the NK-1 receptor indicates that residues involved in non-peptide antagonist binding cluster around the outer portion of transmembrane segments (TM) V and VI. In contrast mutations affecting the binding of the natural peptide agonist, substance P, are scattered in the exterior part of the receptor. Recently it was reported that a number of mutations in TM-II also seriously impair substance P binding. Here we confirm that Ala substitutions for these residues located on a hydrophilic helical face of TM-II basically eliminate substance P binding to the NK-1 receptor, provided that a radiolabeled non-peptide antagonist is used as radioligand. Surprisingly, radiolabeled substance P bound well to all these mutant receptors and was displaced with only slightly reduced affinity by the unlabeled peptide and by the non-peptide antagonists. The wild-type homologous NK-2 receptor displayed properties similar to those observed in the mutated NK-1 receptors, i.e. concomitant high affinity binding of radiolabeled agonist peptide (in this case neurokinin A), yet low affinity, G-protein independent competition of unlabeled peptide with radiolabeled non-peptide antagonist. It is concluded that substitutions in TM-II of the NK-1 receptor do not affect the high affinity binding of substance P but instead block the ability of the peptides to compete for non-peptide antagonist binding. It is suggested that certain mutations can impair interchange between receptor conformations that each bind different ligands with high affinity.
Amino Acid Sequence↗
Specific residues at the top of transmembrane segment V and VI of the neurokinin-1 receptor involved in binding of the nonpeptide antagonist CP 96,345 [corrected].
Previously we have found that binding of the nonpeptide substance P antagonist, CP 96,345, to the neurokinin-1 (NK-1) receptor was critically dependent on two short segments adjacent to the top of transmembrane segments (TM) V and VI, called segments A (residues 183-195) and D (residues 271-276), respectively. In the present study we have systematically performed substitutions of nonconserved residues within these two segments with residues from the homologous NK-3 and/or NK-2 receptor. In segment A, deletion of residues Glu193 and Lys194, which are not present in the NK-3 receptor, or substituting them with leucines as in the NK-2 receptor, decreased the affinity of CP 96,345 10- and 22-fold, respectively. Surprisingly, switching the position of Glu193 and Lys194 did not affect the affinity of CP 96,345, suggesting that, rather than interacting directly with CP 96,345, an interaction of these residues with one another is important for CP 96,345 binding. In segment D substitution of Tyr272 with threonine as in the NK-2 receptor and with alanine as in the NK-3 receptor decreased the affinity of CP 96,345 7- and 24-fold, respectively. Mutation of the preceding Pro271 to glycine alone did not affect CP 96,345 binding, but, combined with the mutation of Tyr272 to threonine, the affinity decreased 28-fold. A series of CP 96,345 analogues with modifications of the major chemical moieties exhibited equally reduced affinity as that of CP 96,345 for the Tyr272- and Lys193-Glu194-substituted constructs, except CP 95,555, which lacks one of the phenyl rings in the benzhydryl group and which was almost unaffected by these mutations. In conclusion, our data indicate a direct interaction between CP 96,345 and Tyr272, which are located at the top of TM VI likely in close spatial proximity to the previously identified interaction point, His197, at the top of the adjacent TM V. Furthermore, the data demonstrated a critical involvement in CP 96,345 binding of Lys193 and Glu194 located one alpha-helical turn above His197.
Amino Acid Sequence↗
The species selectivity of chemically distinct tachykinin nonpeptide antagonists is dependent on common divergent residues of the rat and human neurokinin-1 receptors.
During evolution mutations have occurred in peptide receptors that are neutral with respect to binding of the natural peptide ligand but frequently affect the binding of nonpeptide antagonists. By systematically introducing the nonconserved residues from the human neurokinin (NK)-1 receptor into the corresponding rat receptor we have attempted to localize the structural elements that are responsible for 15-76-fold higher affinity of three tachykinin nonpeptide antagonists for the human receptor, compared with the corresponding rat receptor. Surprisingly, exchange of the four divergent residues located around the previously located apparent binding site for CP 96,345 and FK 888 at the top of transmembrane segment (TM) V and VI, either alone or as a group, did not affect the binding of these nonpeptide compounds. However, substitution of Ser290 in TM VII of the rat receptor with isoleucine present in the human receptor increased the affinity for FK 888 20-fold and that for CP 96345 6-fold, corresponding to an affinity that was only about 4-fold less than the affinity for the human NK-1 receptor. Full human-like affinity for FK 888 and CP 96,345 could be conveyed to the rat receptor by the combined substitution of Ser290 in TM VII to isoleucine and Leu116 in TM III to valine. The NK-2 receptor-selective compound SR 48,968 was found to bind with low affinity to the human NK-1 receptor but with 15-fold even lower affinity to the rat receptor. Substitution of residue 290, which is situated within the previously located binding site for this compound, could completely account for this difference. These data demonstrate that the species selectivities of the nonpeptide antagonists CP 96345, FK 888, and SR 48,968, independently of clear differences in their chemical structures and modes of discovery, have a similar structural basis, being dependent on two divergent residues that apparently are not involved in peptide agonist binding.
Amino Acid Sequence↗
Evidence for a common molecular mode of action for chemically distinct nonpeptide antagonists at the neurokinin-1 (substance P) receptor.
The molecular mechanism of action of three chemically distinct nonpeptide antagonists, SR 140,333, FK 888, and RP 67,580, was compared with that of the previously characterized compound CP 96,345, using a series of chimeric constructs between their common target, the rat neurokinin (NK)-1 (substance P) receptor, and the homologous nonresponsive NK-3 (NKB) receptor. The ability of all four nonpeptide compounds to displace radiolabeled substance P from the NK-1 receptor and their ability to inhibit the peptide-induced increase in inositol phosphate turnover were critically dependent on structural elements located in an area from the middle of the second extracellular loop through transmembrane segments V and VI to the middle of the third extracellular loop of the NK-1 receptor. Dissection of the domain around the outer part of transmembrane segments V and VI into smaller segments demonstrated that the individual nonpeptide antagonists, in agreement with their distinct chemical structures, were dependent on different subepitopes within the common putative binding domain. Full NK-1-like susceptibility to SR 140,333, FK 888, and CP 96,345 could be transferred to the NK-3 receptor by exchange of transmembrane segments V and VI and adjacent parts with corresponding segments from the NK-1 receptor. For SR 140,333 and CP 96,345, almost the same effect could be achieved by transfer of two discontinuous segments around the top of transmembrane segments V and VI. RP 67,580 shared interaction sites with the other compounds around the top of transmembrane segment VI but appeared also to be dependent on transmembrane segment VII. It is concluded that four nonpeptide antagonists, despite overt chemical differences, appear to block NK-1 receptor function by interacting in distinct ways with a common site located spatially around the outer part of transmembrane segment VI.
Animals↗
Conserved HisVI-17 of the NK-1 receptor is involved in binding of non-peptide antagonists but not substance P.
Residue number 17 in transmembrane segment VI has been shown to be crucial for the binding of agonists in G-protein-coupled receptors for the monoamines. In many peptide receptors a histidyl residue has been conserved at this position. We find that replacement of HisVI-17 in the NK-1 receptor with either glutamine, phenylalanine, or alanine has no apparent effect on the binding of the natural peptide ligand substance P or on the agonist induced increase in inositolphosphate turnover. However, the binding of certain non-peptide antagonists was impaired; for example, replacement of HisVI-17 with alanine decreased the affinity for FK888 and RP67,580 5- to 12-fold, respectively. A glutamine side chain was a good substitute for the imidazole in the binding of all non-peptide antagonists. It is concluded that the conserved HisVI-17 in the NK-1 receptor is involved in the binding of certain non-peptide antagonists, but is not important for the action of the natural peptide agonist, substance P.
Amino Acid Sequence↗
A soluble divalent class I major histocompatibility complex molecule inhibits alloreactive T cells at nanomolar concentrations.
Genetically engineered or chemically purified soluble monovalent major histocompatibility complex (MHC) molecules, which have previously been used to study T cells, have not blocked cytotoxic T-cell responses. Here we describe a genetically engineered divalent class I MHC molecule which inhibits lysis of target cells by alloreactive cytotoxic T cells. This protein, H-2Kb/IgG, was generated as a fusion protein between the extracellular domains of a murine class I polypeptide, H-2Kb, and an immunoglobulin heavy chain polypeptide. The chimeric protein has serological and biochemical characteristics of both the MHC and IgG polypeptides. Nanomolar concentrations of H-2Kb/IgG inhibited lysis of H-2Kb-expressing target cells not only by alloreactive H-2Kb-specific T-cell clones but also by alloreactive H-2Kb-specific primary T-cell cultures. A direct binding assay showed high-affinity binding between the H-2Kb/IgG molecule and an H-2Kb-specific alloreactive T-cell clone. Unlabeled H-2Kb/IgG displaced 125I-labeled H-2Kb/IgG from T cells with an IC50 of 1.2 nM.
Amino Acid Sequence↗
Binding epitopes for peptide and non-peptide ligands on the NK1 (substance P) receptor.
Explore the source record for details and available documents.
Amino Acid Sequence↗
Two nonpeptide tachykinin antagonists act through epitopes on corresponding segments of the NK1 and NK2 receptors.
The molecular mechanism of action for two chemically distinct and highly selective, nonpeptide antagonists, CP-96,345 and SR-48,968, was studied by development of a series of chimeric constructs between their respective target receptors, the NK1 (substance P) and NK2 (neurokinin A) receptors. The binding affinities of the natural peptide ligands, substance P and neurokinin A, were not affected by exchanging almost the entire C-terminal half of the NK1 receptor with the corresponding segment of the NK2 receptor. In contrast, it was found that transfer from the NK2 to the NK1 receptor of a segment corresponding to transmembrane segment VI, the amino-terminal half of transmembrane segment VII, and the connecting extracellular loop 3 completely switched the susceptibility for the nonpeptide antagonists. This chimeric exchange, corresponding to 17 nonconserved residues, conveyed full susceptibility for the NK2-specific compound SR-48,968 to the previously unresponsive NK1 receptor--i.e., the Ki value for inhibition of binding of 125I-labeled substance P decreased from > 10,000 to 0.97 nM. At the same time the affinity for the NK1-selective compound CP-96,345 decreased > 30-fold. The actual binding site for SR-48,968 was localized to this region of the NK2 receptor by use of [3H]SR-48,968, which did not bind to the NK1 receptor but bound with similar high affinities to the wild-type NK2 receptor and to the chimeric NK1 receptor with the NK2 receptor segment incorporated around transmembrane segments VI and VII, Kd = 1.5 nM and 1.0 nM, respectively. Our data indicate that two chemically very different nonpeptide antagonists, CP-96,345 and SR-48,968, act through epitopes located around transmembrane segment VI on their respective target receptors and that at least the nonconserved residues in these epitopes are not important for the binding of the natural peptide ligands, substance P and neurokinin A.
Animals↗