PubMed Health⌕ Search

Biomedical subjects

M Rola-Pleszczynski

Publications and source records attributed to M Rola-Pleszczynski.

At least 19 recordsLinked to original sources
PubMed1999

Selective modulation of wild type receptor functions by mutants of G-protein-coupled receptors.

Members of the G-protein-coupled receptor (GPCR) family are involved in most aspects of higher eukaryote biology, and mutations in their coding sequence have been linked to several diseases. In the present study, we report that mutant GPCR can affect the functional properties of the co-expressed wild type (WT) receptor. Mutants of the human platelet-activating factor receptor that fail to show any detectable ligand binding (N285I and K298stop) or coupling to a G-protein (D63N, D289A, and Y293A) were co-expressed with the WT receptor in Chinese hamster ovary and COS-7 cells. In this context, N285I and K298stop mutant receptors inhibited 3H-WEB2086 binding and surface expression. Co-transfection with D63N resulted in a constitutively active receptor phenotype. Platelet-activating factor-induced inositol phosphate production in cells transfected with a 1:1 ratio of WT:D63N was higher than with the WT cDNA alone but was abolished with a 1:3 ratio. We confirmed that these findings could be extended to other GPCRs by showing that co-expression of the WT C-C chemokine receptor 2b with a carboxyl-terminal deletion mutant (K311stop), resulted in a decreased affinity and responsiveness to MCP-1. A better understanding of this phenomenon could lead to important tools for the prevention or treatment of certain diseases.

Animals↗
PubMed1999

Modulation of formyl peptide receptor expression by IL-10 in human monocytes and neutrophils.

IL-10, originally described as a cytokine synthesis inhibitory factor, is secreted by a number of cells of the immune system, including monocytes and T cells. Although IL-10 is being assigned as an immunosuppressive cytokine, our study showed that FMLP-R mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of this cytokine, with maximal (three- to fourfold) stimulation with 10 ng/ml. The effect was rapid, being observable as early as 1 h of treatment with IL-10, maximal between 2 and 4 h, and still evident after 24 h and was associated with an increase of receptor expression on the cell surface as assessed by flow cytometry analysis. Pretreatment of monocytes with actinomycin D completely abrogated the effect of IL-10, suggesting a transcriptional regulation. Moreover, IL-10-treated monocytes showed a significantly enhanced functional responsiveness to FMLP with enhanced (three- to fourfold) chemotaxis and augmented (twofold) intracellular calcium mobilization. In polymorphonuclear neutrophils (PMN), IL-10 also mediated a twofold augmentation of FMLP-R expression. In parallel experiments, we observed that IL-10 could differentially modulate other chemotactic receptors. Hence, we observed that IL-10 augmented two-to threefold platelet-activating factor receptor (PAF-R) expression, whereas it had no significant effect on the fifth component of complement (C5a) receptor (C5a-R) expression. Collectively, our results demonstrate that IL-10 may play an important role in inflammatory process through modulation of chemotactic receptor expression.

Chemotaxis, Leukocyte↗
PubMed1999

IL-10 up-regulates CCR5 gene expression in human monocytes.

The chemokine receptor CCR5 has been found to play a key role in early infection with macrophage-tropic isolates of HIV-1 and CCR5 deficiency is associated with a relative resistance to HIV-1 infection. In this context, we studied the regulation of CCR5 gene expression by cytokines, and in particular, interleukin (IL)-10 in human monocytes. CCR5 mRNA was rapidly up-regulated by exposure of monocytes to graded concentrations of IL-10, with near maximal stimulation with 10 ng/ml. The effect was rapid, being detectable as early as 1 h and maximal between 2 and 4 h of treatment. Pretreatment of monocytes with actinomycin D prevented the IL-10-induced effect, suggesting a transcriptional mechanism for CCR5 up-regulation by IL-10. Protein expression of CCR5 was also enhanced by IL-10, as indicated by a 3-fold increase in anti-CCR5 antibody labeling of monocytes treated for 20 h with IL-10. Increased surface expression of CCR5 persisted at 48 h of treatment. Moreover, IL-10-treated monocytes responded with augmented intracellular Ca++ mobilization and enhanced (3-4-fold) chemotaxis in response to the CCR5 ligand MIP-1beta(25 ng/ml). Taken together, our data indicate that IL-10 can modulate CCR5 expression and function. This can constitute a potentially important regulatory mechanism which can affect not only responses during inflammation, but also susceptibility to HIV-1 infection.

Antibodies, Monoclonal↗
PubMed1998

Signalling through the leukotriene B4 receptor involves both alphai and alpha16, but not alphaq or alpha11 G-protein subunits.

COS-7 cells transfected with the leukotriene (LT) B4 receptor (BLTR) cDNA were unable to produce LTB4-induced inositol phosphates (IPs) in spite of the presence of endogenous Galphai, Galphaq and Galpha11 proteins. Co-transfection of BLTR with Galpha16, however, resulted in high levels of IP production, which were 17-, 10- and 6-fold higher than with co-transfected Galpha11, Galphaq and Galpha14, respectively. Co-transfection of BLTR with phospholipase C (PLC) beta2, on the other hand, resulted in efficient IP production and co-transfection of BLTR with both Galpha16 and PLCbeta2 resulted in a greater than additive response.

Animals↗
PubMed1998

Prostaglandin E2 induces resistance to human immunodeficiency virus-1 infection in monocyte-derived macrophages: downregulation of CCR5 expression by cyclic adenosine monophosphate.

The chemokine receptor CCR5 can function as a coreceptor for human immunodeficiency virus-1 (HIV-1) entry into CD4(+) T cells and macrophages, especially during the early stages of HIV-1 infection. The regulation of CCR5 expression may affect not only leukocyte migration, but also infectivity by HIV-1 and, therefore, acquired immunodeficiency syndrome (AIDS) pathogenesis. We report here that agents which increase intracellular concentrations of cyclic adenosine monophosphate (cAMP) rapidly downregulate CCR5 gene expression, with consequent loss of CCR5 expression and function in monocytes/macrophages. Chemotaxis and intracellular Ca2+ mobilization in monocytes pretreated with prostaglandin E2 or dibutyryl-cAMP for 24 hours were significantly reduced in response to the CCR5 ligand, MIP-1beta. Moreover, HIV-1 entry into monocyte-derived macrophages pretreated with dibutyryl-cAMP or prostaglandin E2 was markedly decreased. Our findings suggest that resistance to HIV-1 can be induced by agents which increase cellular levels of cAMP and that this may suggest additional therapeutic strategies to limit infection by HIV-1.

Cyclic AMP↗
PubMed1998

Localization, quantification, and activation of platelet-activating factor receptor in human endometrium during the menstrual cycle: PAF stimulates NO, VEGF, and FAKpp125.

Implantation is characterized by an inflammatory-like response with expansion of extracellular fluid volume, increased vascular permeability, and vasodilatation. These effects are believed to be mediated at the paracrine level by prostaglandin E2 and platelet-activating factor (PAF), but the cellular mechanism (or mechanisms) remains largely unknown. We demonstrate that PAF receptor (PAF-R) immunoreactivity and mRNA are detected in proliferative and secretory endometrial glands, however, the responsiveness of endometrium to physiological concentrations of PAF is confined predominantly to the secretory endometrium. Semiquantitative reverse transcription-polymerase chain reaction revealed that PAF-R transcript levels were highest in the mid-late proliferative and late secretory phases of the cycle. Interaction of PAF with its receptor resulted in the rapid release of nitric oxide (NO), increased expression of vascular endothelial growth factor (VEGF), and activation of FAKpp125, a focal adhesion kinase, demonstrating that the PAF-R is functionally active. Inhibition of NO synthesis by NG-monomethyl-L-arginine produced dose-dependent attenuation of PAF-evoked NO release, indicating NOS activation; the dependency of PAF-evoked NO release on PKC and extracellular Ca2+ was confirmed by PKC inhibitor Ro 31-8220 and by the removal of extracellular Ca2+. PAF up-regulated VEGF gene expression in a concentration- and time-dependent fashion in human endometrial epithelial cell lysates. Transcription of VEGF was rapidly followed by secretion of the protein. These data support our premise that this autocoid acts as an angiogenic mediator in the regeneration of the endometrium after menses and as a vasodilator to promote blastocyst attachment during the implantation process.

Cell Adhesion Molecules↗
PubMed1997

Epstein-Barr virus induces GM-CSF synthesis by monocytes: effect on EBV-induced IL-1 and IL-1 receptor antagonist production in neutrophils.

Neutrophils play an important role in the control of viral infections by releasing a variety of potent agents. We previously demonstrated that Epstein-Barr virus (EBV) binds to human neutrophils and stimulates cytokine synthesis including interleukin-1 (IL-1) and IL-1 receptor antagonist (IL-1Ra). Since neutrophil functions are known to be modulated by the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF), we therefore investigated the cellular source of GM-CSF synthesis following treatment of leukocytes with EBV and the effect of GM-CSF on the production of IL-1, IL-1Ra, and superoxide by EBV-treated neutrophils. In enriched-cell populations, only monocytes were found to produce GM-CSF in response to EBV, which was maximal after 12 h of incubation. The results obtained with UV-irradiated particles or EBV neutralized with monoclonal antibody 72A1 suggest that contact between the cell and the gp350 of the viral envelope is sufficient to induce the release of GM-CSF. On the other hand, GM-CSF differentially upregulated EBV-induced IL-1 and IL-1Ra production by neutrophils. Pretreatment of neutrophils with GM-CSF prior to EBV activation synergistically enhanced the production of IL-1 alpha and IL-1 beta, but only marginally affected IL-1Ra synthesis. In addition, GM-CSF was also found to synergistically enhance the superoxide production by neutrophils in response to EBV. Molecular analysis showed that GM-CSF did not alter the IL-1 beta and IL-1Ra mRNA synthesis induced by EBV, suggesting that GM-CSF could act at a posttranslational level. Local production of GM-CSF by monocytes in tissues invaded by EBV could serve to potentiate the host defense mechanisms directed toward the destruction of the infectious virus.

Animals↗
PubMed1997

Structural and functional requirements for agonist-induced internalization of the human platelet-activating factor receptor.

The receptor for platelet-activating factor (PAF) is a member of the G-protein-coupled receptor family. To study the structural elements and mechanisms involved in the internalization of human PAF receptor (hPAFR), we used the following mutants: a truncated mutant in the C-terminal tail of the receptor (Cys317 --> Stop) and mutations in the (D/N)P(X)2,3Y motif (Asp289 --> Asn,Ala and Tyr293 --> Phe,Ala). Chinese hamster ovary cells expressing the Cys317 --> Stop mutant exhibited a marked reduction in their capacity to internalize PAF, suggesting the existence of determinants important for endocytosis in the last 26 amino acids of the cytoplasmic tail. Substitution of Asp289 to alanine abolished both internalization and G-protein coupling, whereas substitution of Tyr293 to alanine abolished coupling but not internalization. Inhibition or activation of protein kinase C did not significantly affect the internalization process. Receptor sequestration and ligand uptake was, at least in part, blocked by concanavalin A and blockers of endocytosis mediated by clathrin-coated pits. Our data suggest that the internalization of a G-protein-coupled receptor and coupling to a G-protein can be two independent events. Moreover, the C terminus tail of hPAFR, but not the putative internalization motifs, may be involved in the internalization of hPAFR.

Animals↗
PubMed1997

Role of the Cys90, Cys95 and Cys173 residues in the structure and function of the human platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent phospholipid mediator which binds to a specific, high affinity receptor of the G protein-coupled receptor family. In the present report, we show that ligand binding to the PAF receptor is sensitive to the reducing agent dithiothreitol (DTT), suggesting the involvement of disulfide linkages in the proper PAF receptor conformation. Substitutions of Cys90, Cys95 and Cys173 to Ala or Ser demonstrated that these cysteine residues are critical for normal cell surface expression of the PAF receptor protein and ligand binding to the receptor. The Cys90 and Cys173 mutant receptors did not display any specific ligand binding, were not expressed on the cell surface but were found in the intracellular compartment. The Cys95 mutants showed specific binding and were able to stimulate low levels of inositol phosphate (IP) production. These mutants were expressed at low density on the cell surface and showed high expression intracellularly. Our results suggest that the structure and function of the PAF receptor require the conserved Cys90 and Cys173 to form a disulfide bond. Moreover, Cys95 also appears to be necessary, possibly by establishing a disulfide linkage with an as yet unidentified Cys residue. All three residues appear essential for the proper folding and surface expression of the PAF receptor protein.

Amino Acid Sequence↗
PubMed1997

Augmented expression of platelet-activating factor receptor gene by TNF-alpha through transcriptional activation in human monocytes.

Tumor necrosis factor alpha (TNF-alpha) is a cytokine produced by activated monocytes and often associated with platelet-activating factor (PAF) during the pathogenesis of many inflammatory and infectious diseases. PAFR is a G-protein-coupled receptor constitutively expressed on monocytes. TNF-alpha (100-400 U/mL) significantly increased PAFR mRNA expression in human monocytes. This increase was seen after 1 h of stimulation and persisted up to 24 h. Actinomycin D pretreatment studies revealed a transcriptional increase in PAFR gene expression without effect on mRNA half-life. [3H]WEB 2086 binding studies showed a significant (43%) increase in specific binding sites in 24-h-treated cells without change in receptor affinity. Increased interleukin-6 production in response to PAF was also found in 24-h TNF-alpha-pretreated monocytes. These observations provide new evidence for TNF-alpha and PAF interactions in human monocytes during inflammatory processes through up-regulation of PAFR expression by TNF-alpha.

Azepines↗
PubMed1997

Changes in apoptosis of human polymorphonuclear granulocytes with aging.

Many alterations with aging occur at the cellular and organic levels in the immune system ultimately leading to a decrease in the immune response. Our aim in the present work was to study apoptosis of polymorphonuclear granulocytes (PMN) with aging under various stimulations since apoptosis might play an important role in several pathologies encountered with aging. The PMN of healthy young (20-25 years) and elderly (65-85 years) subjects were examined after 24 h of sterile culture with and without stimulation. The stimulating agents included: phorbol myristate acetate (PMA), hydrogen peroxide (H2O2), N-formyl-methionyl-leucyl phenylalanine (FMLP), granulocyte-macrophage colony stimulating factor (GM-CSF), reduced glutathione (GSH), lipopolysaccharide (LPS) and interleukin 2 (IL-2). Apoptosis was assessed by traditional staining of the plates, by flow cytometric staining and DNA gel electrophoresis. It was found that without stimulation the susceptibility of PMN to apoptosis was slightly increased with aging. Under various stimulations, such as PMA. H2O2, apoptosis was almost 100%, while the treatment by FMLP, oxLDL and GSH did not change its extent in PMN obtained either from young or elderly subjects. Marked age-related changes were observed in the extent of apoptosis under stimulation with GM-CSF, IL-2 and LPS. These agents were able to significantly prevent apoptosis in PMN of young subjects, while only the GM-CSF was able to slightly modulate it in neutrophils of elderly subjects. From these results, we suggest that changes in apoptosis of PMN with aging could play a role in the increased incidence of certain immune system related pathologies of aging, such as cancer, infections and autoimmune disorders.

Adult↗
PubMed1997

Platelet-activating factor stimulates interleukin-6 production by human endothelial cells and synergizes with tumor necrosis factor for enhanced production of granulocyte-macrophage colony stimulating factor.

The interaction between human endothelial cells (EC) and leukocytes during inflammation is in part mediated through the release of soluble factors. Since platelet-activating factor (PAF) is a potent mediator of inflammatory responses, we investigated the potential of PAF to modulate IL-6 and GM-CSF production by EC. Exposure of these cells to PAF resulted in a concentration-dependent increase in IL-6 production, with a maximum at 10(-10) M PAF. Sequential incubation of EC with PAF and TNF alpha resulted in a synergistic increase of IL-6 production. This effect was specific for PAF since it was prevented by preincubation with the PAF receptor antagonist, WEB 2086. Northern blot analysis revealed enhanced IL-6 mRNA expression in PAF-treated EC. However, the synergy observed in protein synthesis between PAF and TNF alpha was not reflected in IL-6 mRNA accumulation, suggesting a post-translational modulation. Pretreatment of EC with the protein synthesis inhibitor cycloheximide before their exposure to PAF resulted, after washout of the cycloheximide, in a markedly augmented production of IL-6, suggesting a synergy between augmented IL-6 mRNA accumulation by PAF and IL-6 mRNA superinduction by cycloheximide. GM-CSF production by EC was also stimulated by the combined effects of PAF and TNF alpha, but PAF alone did not affect GM-CSF production. Taken together, our data suggest that PAF can stimulate EC to synthesize cytokines, including IL-6 and GM-CSF, which may contribute to local and, possibly, systemic responses during inflammation.

Azepines↗
PubMed1997

Differential signaling pathways in platelet-activating factor-induced proliferation and interleukin-6 production by rat vascular smooth muscle cells.

Vascular smooth muscle cells (SMCs) can be induced to proliferate in response to several cytokines and growth factors, including interleukin (IL)-6. Platelet-activating factor (PAF) also has been shown to induce SMC proliferation. Because PAF can stimulate IL-6 production in monocytes, macrophages, and endothelial cells, our study was undertaken to determine whether PAF could induce IL-6 production by SMCs and to define the underlying signaling pathways. Exposure of rat aortic SMCs to picomolar concentrations of PAF resulted in enhanced production of IL-6. The effect was concentration dependent, selective for the active form of PAF, and mediated by specific PAF receptors. Pretreatment of the cells with Bordatella pertussis toxin (PTX) prevented the effect of PAF, suggesting the involvement of alpha i-type subunits of G proteins in the signal-transduction pathway. PAF-dependent IL-6 production was also prevented by inhibition of tyrosine kinases with genistein or erbstatin. Inhibition of eicosanoid production by blocking either phospholipase A2 or cyclooxygenase also abrogated the effect of PAF on IL-6 production. Moreover, inhibition of Ca2+-calmodulin activity with W7 or blocking of calcium channels with verapamil or nifedipine prevented PAF-mediated enhancement of IL-6 production. Whereas PAF-induced signal-transduction pathways leading to IL-6 production and SMC proliferation were partially common, they appeared to diverge downstream of PLA2 activation: inhibition of cyclooxygenase had no effect on proliferation, whereas augmentation of cyclic adenosine monophosphate (cAMP) levels or activation of protein kinase A inhibited proliferation, in contrast to IL-6 production. Our findings suggest a role for PAF in modulating vascular function by stimulating local production of IL-6 by SMCs and promoting their proliferation. The two effects are, however, associated with partially divergent signaling pathways and may not be causally related.

Animals↗
PubMed1997

Functional IL-2 receptors are expressed by rat lung type II epithelial cells.

Interferon (IFN)-gamma-induced inhibition of type II epithelial cell thymidine incorporation (45% decrease vs. control) was restored by cocultures with mitogen-activated peripheral blood mononuclear cells (PBMC) and conditioned media (CM) from mitogen-activated PBMC. Successive exposure of type II cells to IFN-gamma and interleukin (IL)-2 produced similar alterations in thymidine incorporation. Given that IL-2 is a powerful pleiotropic cytokine produced by lymphoid and myeloid cells, the presence of IL-2 receptors (IL-2R) was assessed in primary cultures of rat type II pneumocytes (TIIP) and the nontransformed alveolar type II epithelial cell line L2. The presence of IL-2R membrane protein on rat type II cells was established by immunodetection assays. The expression of all three murine IL-2R alpha-, beta-, and gamma-chain RNA transcripts in primary TIIP cultures and L2 cells was highlighted by reverse transcriptase-polymerase chain reaction analysis. Overall, these experiments demonstrate, for the first time, that type II epithelial cells can express functional IL-2R, confirming TIIP as a potential "partner" in the lung immune system. Consequently, it can be speculated that TIIP are new cellular targets for lymphokines using (IL-2R) gamma-chain-bearing receptors in lung distal air-spaces.

Animals↗
PubMed1996

Modulation of human platelet-activating factor receptor gene expression by protein kinase C activation.

The effect of PMA on early and late regulation of platelet-activating factor receptor (PAF-R) expression was examined in human monocytes. Treatment with 16 nM PMA for 5 min initiated a rapid reduction (50-75%) in [3H]WEB 2086 binding, which was maximal between 5 and 15 min. Scatchard analysis revealed that PMA treatment reduced the number of binding sites to 50% of control cells without an appreciable change in their affinity. In parallel cultures, flow cytometry analysis using anti-PAF-R Abs failed to reveal any significant decrease in the level of PAF-R expression until after 4 h of treatment with PMA. By 24 h, PAF-R expression had declined by 80 to 90%. This PMA-induced down-regulation of surface PAF-R expression was preceded by a rapid down-regulation of PAF-R mRNA expression. PMA-mediated down-regulation could be blocked by the protein kinase C (PKC) inhibitors H-7 and calphostin C. Activation of PKC by the diacylglycerol analogue 1-oleoyl-2-acetylglycerol also resulted in down-regulation of PAF-R mRNA accumulation, whereas the inactive phorbol diester 4alpha-PMA was ineffective. The rapid disappearance of the PAF-R transcripts was associated with decreased stability of receptor mRNA and not with a change in the nuclear transcription rate of the PAF-R gene. These findings indicate that PAF-R gene expression in human leukocytes can be regulated through a PKC-dependent pathway and involves post-transcriptional destabilization of receptor mRNA.

Enzyme Activation↗
PubMed1996

Identification of transmembrane domain residues determinant in the structure-function relationship of the human platelet-activating factor receptor by site-directed mutagenesis.

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.

Amino Acid Sequence↗
PubMed1996

Mutations of two adjacent amino acids generate inactive and constitutively active forms of the human platelet-activating factor receptor.

We have mutated two residues, Ala230 and Leu231, in the C-terminal portion of the third intracellular loop of the human platelet-activating factor (PAF) receptor into Glu230 and Arg231, respectively. The Leu231 --> Arg231 substitution led to two major modifications: 1) increased constitutive activity of the PAF receptor resulting in agonist-independent production of inositol phosphates and 2) increased affinity of the receptor for binding PAF (agonist) but not WEB2086 (antagonist). The L231R mutant was able to adopt at least two conformations: (i) a higher affinity state than the corresponding state of the wild-type receptor (WT), dependent on G protein coupling, and (ii) a low affinity state, higher than the one for the uncoupled WT receptor. The Ala230 --> Glu230 substitution also resulted in two major modifications: 1) unresponsiveness in terms of phosphatidylinositol hydrolysis in response to PAF and 2) a marked decrease in affinity of the receptor for binding the agonist but not the antagonist. Competition binding studies of transient receptor expression in COS-7 cells and the inability of guanosine 5 -O-(3-thiotriphosphate) to modulate the decrease in affinity of a stable A230E mutant in Chinese hamster ovary cells suggest an inherent low affinity conformation for this mutant. Alternatively, mutation of Ala230 to Gln230 suggested that the residue 230 has a fundamental effect on receptor affinity and its charge is determinant in G protein coupling of the PAF receptor. In this report, we show that substitution of two immediately adjacent residues of the PAF receptor, Ala230 and Leu231, surprisingly leads to an inactive and a constitutively active phenotype, respectively. These results further support the concept of constitutively active G protein-coupled receptors as adopting ''active'' state conformations similar to those induced by agonist binding to WT receptors.

Amino Acid Sequence↗