RESUMEN
A major hurdle in the structural analysis of membrane proteins is the expression of a functional and homogeneous form of the protein. Except for rhodopsin, most G protein-coupled receptors (GPCRs) are endogenously expressed at very low levels. Heterologous expression of GPCRs in bacteria, yeast, insect cells or mammalian cell lines often yields proteins with large amounts of misfolded proteins and heterogeneous posttranslational modifications. Here, we report a novel mammalian "in vivo" system for the expression of the chemokine receptor CXCR1. This receptor was expressed in liver of mice infected with adenovirus encoding CXCR1. Liver plasma membranes from infected mice displayed high-levels of (125)I-labeled human interleukin-8 (IL-8) binding. The pharmacological profile of the recombinant CXCR1 expressed "in vivo" was similar to those expressed in neutrophils. We found that the incorporation of the detergent solubilized CXCR1 into phospholipid vesicles in the presence of Gi/Go proteins is required for the reconstitution of (125)I-IL-8 binding. On the basis of the presence of the several endogenous His residues and glycosylation moieties in CXCR1 we fractionated the detergent-solubilized plasma membranes by employing Ni- and Concanavalin A-based chromatography. Fractions enriched with CXCR1 were monitored by (125)I-IL-8-bound to the receptor and Western blots with anti-CXCR1 antibodies. This robust expression system could be readily applied for the expression of GPCRs and other eukaryotic membrane proteins.
Asunto(s)
Adenoviridae/genética , Receptores de Interleucina-8A/metabolismo , Adenoviridae/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Células Cultivadas , Humanos , Interleucina-8/genética , Interleucina-8/metabolismo , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Datos de Secuencia Molecular , Conejos , Receptores de Interleucina-8A/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.
Asunto(s)
Radical Hidroxilo , Receptores de Angiotensina/química , Receptores de Angiotensina/genética , Receptores de Bradiquinina/química , Receptores de Bradiquinina/genética , Transducción de Señal , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Línea Celular , Cartilla de ADN/farmacología , Endocitosis , Microscopía Fluorescente , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fosfatidilinositoles/metabolismo , Estructura Terciaria de Proteína , Ratas , Receptor de Bradiquinina B2 , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Factores de Tiempo , TransfecciónRESUMEN
Rapid induction and down-regulation of bradykinin B1 receptor (BKB1R) gene expression is tightly regulated at the transcriptional and mRNA levels (Zhou et al. [1998] Biochem. J. 330:361-366; Zhou et al. [1999] Mol. Cell Biol. Res. Commun. 1:29-35). Here we explore regulation of BKB1R expression at the protein level. To make this inducible gene express constitutively, we utilized a bicistronic mammalian expression vector (pCMin) for stable transfection of the BKB1R gene into human lung fibroblasts, IMR90SV40. The BKB1R displayed a high affinity and specificity (K(d) = 0.5 nM) for desArg(10)-kallidin. The receptor mediated such signaling events as arachidonic acid (ARA) release, phosphoinositide (PI) turnover and Ca(2+)-flux. The receptor function proved differentially desensitized. For example, after initial exposure to desArg(10)-kallidin, a second stimulation with desArg(10)-kallidin did not induce further Ca(2+)-flux or ARA-release while PI-turnover continued unabated. Unlike most of the G-protein coupled receptors, the BKB1R did not internalize within 60 min of exposure to 10 nM desArg(10)-kallidin. It also did not resensitize. Thus, the duration and signal capacity of the BKB1R at the protein level is regulated through lack of internalization, an absence of resensitization and a lack of desensitization for certain events such as PI turnover. In fact, the absence of BKB1R resensitization is likely a very important contributor to the rapid disappearance of this inducible receptor.
Asunto(s)
Endocitosis , Regulación de la Expresión Génica , Calidina/análogos & derivados , Pulmón/metabolismo , Receptores de Bradiquinina/biosíntesis , Receptores de Bradiquinina/genética , Ácido Araquidónico/metabolismo , Northern Blotting , Bradiquinina/farmacología , Calcio/metabolismo , Células Cultivadas , Cartilla de ADN/química , Fibroblastos/metabolismo , Vectores Genéticos , Humanos , Calidina/farmacología , Pulmón/citología , Fosfatidilinositoles/metabolismo , Receptor de Bradiquinina B1 , Receptor de Bradiquinina B2 , TransfecciónRESUMEN
We showed previously that the inducible bradykinin B1 receptor (BKB1R) gene expression is regulated, in part, through mRNA stabilization. Here we clone the 3'-untranslated region (3'-UTR) of the BKB1R. This region proves to be very short, containing only 14 bases with an alternative polyadenylation signal (AUUAAA) which overlaps with the stop codon. Reverse transcription confirms the presence of this alternative polyadenylation signal. Northern blot shows a single species of BKB1R mRNA of approximately 1.4 kb in agreement with its calculated length. The BKB1R mRNA induced by TNFalpha, phorbol ester, bradykinin, and desArg10-kallidin contain the same 3'-UTR species. To test the role of this region in the regulation of mRNA stability, we generated a chimeric luciferase construct containing the BKB1R 3'-UTR. The mRNA transcribed from the wild-type luciferase gene displayed a half-life of approximately 6 h. The mRNA transcribed from the chimeric construct displayed a half-life of only 1 h. This decrease was also reflected at the level of enzyme activity. Luciferase activity from cells transfected with the chimeric construct was 10 times less than from cells transfected with wild-type luciferase. The data presented provide compelling evidence that the 3'-UTR is participating in the regulation of BKB1R mRNA stability and its ultimate expression.
Asunto(s)
ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Bradiquinina/genética , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular , Clonación Molecular , Cartilla de ADN/genética , Regulación de la Expresión Génica , Humanos , Técnicas In Vitro , Luciferasas/genética , Datos de Secuencia Molecular , Procesamiento Postranscripcional del ARN , Estabilidad del ARN , Ratas , Receptor de Bradiquinina B1 , Proteínas Recombinantes de Fusión/genética , TransfecciónRESUMEN
In the search for the structural elements participating in signal transduction, internalization, and resensitization of the bradykinin B2 receptor, we identified two critical motifs, one in the second intracellular loop (IC2), the other in the proximal C terminus. We previously described the contribution of tyrosines within each of the two motifs (Tyr131 and Tyr322) to signal transduction and receptor internalization (Prado, G. N., Taylor, L., and Polgar, P. (1997) J. Biol. Chem. 272, 14638-14642). Here, we investigate the effect of exchanging both tyrosine residues simultaneously for alanine, phenylalanine, or serine, termed YAYA (Y131A/Y322A), YFYF (Y131F/Y322F), and YSYS (Y131S/Y322S) receptors, respectively. All of these mutants bound bradykinin (BK) normally, with a Kd of approximately 1.1 nM. However, although phosphoinositide (PI) turnover in response to BK by Y131A and Y131S proved negligible, the YAYA mutant returned BK-activated PI turnover to wild type (WT). In contrast, PI turnover with YSYS remained unresponsive to BK. Importantly, the pattern of BK-activated arachidonate release differed markedly in the mutant receptors. For example, whereas Y131S ablated BK-activated arachidonic acid release, conversion of this mutant to YSYS returned the BK-activated receptor function to a level above that of WT. However, YAYA showed only a partial recovery from the poor BK response of Y131A. These and additional results suggest that Tyr131 and Tyr322 interact cooperatively in conjunction with at least two separate signaling functions. Given these results, a molecular model of the receptor was generated with the IC2 and the proximal C terminus in close spatial proximity. Conformations were identified to provide structural explanation for these observations. The conserved Thr137 in the IC2 was next substituted with proline (T137P) to prevent phosphorylation at this position or with aspartate (T137D) to emulate phosphorylation. The T137P mutant demonstrated no change from WT with respect to either BK-activated PI turnover or arachidonic acid release. However, the mutant exhibited a markedly reduced capacity to internalize. It also resensitized poorly. The T137D mutant lacked both BK responsive activities. However, it internalized and resensitized normally, as did WT. These final results suggest that Thr137 is functioning as a switch in termination of signal transduction and the initiation of internalization.
Asunto(s)
Endocitosis/genética , Receptores de Bradiquinina/metabolismo , Transducción de Señal/genética , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Línea Celular , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fosfatidilinositoles/metabolismo , Conformación Proteica , Ratas , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/química , Receptores de Bradiquinina/genética , Tirosina/genética , Tirosina/metabolismoRESUMEN
Presently, little is known of the amino acid motif(s) participating in bradykinin B2 receptor-mediated signal transduction processes. In this report we investigate the potential role of the two existing tyrosine (Tyr) residues in the intracellular regions and the carboxyl terminus in the regulatory function of this receptor. Rat-1 cells, which do not contain detectable bradykinin B2 receptor, were transfected with wild type and mutant receptor cDNAs. Tyr-131 and Tyr-321 were each mutated to corresponding alanine-, serine-, and phenylalanine-containing sequences. The last 34 amino acid residues of the carboxyl terminus were truncated. Rat-1 cells transfected with the mutant forms of the receptor cDNA including the truncated COOH-terminal cDNA all bound [3H]bradykinin with essentially the same Kd of approximately 2.2 nM as cells transfected with the wild type bradykinin B2 receptor. However, mutating Tyr-131 resulted in important changes in bradykinin-stimulated phosphoinositide turnover and arachidonate release. For example, exchanging Tyr-131 for alanine led to an 80% decreased arachidonate release (p < 0.005), 90% decrease in inositol phosphate (IP) accumulation (p < 0.001), with receptor uptake at 15 min remaining essentially unchanged. Mutating the same Tyr to phenylalanine resulted in unchanged bradykinin-stimulated IP accumulation, only a slightly lowered arachidonate release, and unchanged receptor uptake at 15 min. Mutating Tyr-321 to alanine resulted in a very different pattern. There was a small but significant reduction in arachidonate release (p < 0.03) and IP accumulation (p < 0.008) with a large, 30%, increase in receptor uptake at 15 min (p < 0.010). Truncation of a portion of the carboxyl tail also proved meaningful, with a 60% decrease in arachidonate release and an 80% decrease in IP accumulation. The truncation also resulted in a large, 130%, decrease in receptor uptake at 15 min (p < 0.023). Taken together, these results point to Tyr-131 as an important element in determining bradykinin-stimulated arachidonate release and IP accumulation. Tyrosine phosphorylation at this site apparently does not play a major role. Tyr-131, Tyr-321, and the carboxyl tail appear to be important in determining receptor uptake.
Asunto(s)
Bradiquinina/farmacología , Estructura Secundaria de Proteína , Receptores de Bradiquinina/química , Receptores de Bradiquinina/fisiología , Tirosina , Secuencia de Aminoácidos , Animales , Ácido Araquidónico/metabolismo , Secuencia Conservada , Endocitosis , Fosfatos de Inositol/metabolismo , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Ratas , Receptor de Bradiquinina B2 , Receptores de Bradiquinina/biosíntesis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Eliminación de Secuencia , Transducción de SeñalRESUMEN
Interleukin 8 (IL-8) is a potent neutrophil chemoattractant and activator. Two IL-8 receptor subtypes, A and B, are expressed in neutrophils. In this work, we analyzed the role of the C terminus domain of the IL-8 receptor on the signal transduction and receptor internalization mechanisms. The IL-8 receptor A was tagged with an epitope corresponding to the monoclonal antibody 1D4 to monitor the localization of the IL-8 receptor. We demonstrated IL-8-dependent receptor internalization by monitoring the density of surface 125I-labeled IL-8 binding sites and by immunofluorescence microscopy. Truncation of the last 27 amino acids of the IL-8 receptor A severely impaired the IL-8-induced internalization of the receptor. Of importance was the observation that binding of IL-8 to receptors A and B triggered a dramatically faster rate of internalization of receptor B than receptor A, suggesting that the heterologous C termini among receptor subtypes modulate the rate of internalization of IL-8 receptors. However, substitution of the C terminus of the receptor subtype A for the C terminus of receptor B reduced the internalization rate of receptor A. Furthermore, we found that the rate of internalization of IL-8 receptor B triggered by IL-8 was faster than the one induced by the IL-8-related peptide, melanoma growth stimulatory activity. Studies with human neutrophils pretreated with 100 nM IL-8 for 5 min revealed a positive and a negative calcium response mediated by receptors A and B, respectively. In contrast, neutrophils pretreated with melanoma growth stimulatory activity showed positive calcium responses to both receptors A and B. These data suggest that the neutrophil responses mediated by IL-8 are modulated by the rate of internalization of receptors.
Asunto(s)
Antígenos CD/metabolismo , Quimiocinas CXC , Péptidos y Proteínas de Señalización Intercelular , Receptores de Interleucina/metabolismo , Transducción de Señal , Animales , Antígenos CD/genética , Antígenos CD/inmunología , Secuencia de Bases , Células CHO , Calcio/metabolismo , Quimiocina CXCL1 , Factores Quimiotácticos/farmacología , Clonación Molecular , Cricetinae , ADN Complementario , Epítopos/inmunología , Sustancias de Crecimiento/farmacología , Humanos , Interleucina-8/metabolismo , Interleucina-8/farmacología , Datos de Secuencia Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Ensayo de Unión Radioligante , Receptores de Interleucina/genética , Receptores de Interleucina/inmunología , Receptores de Interleucina-8ARESUMEN
Interleukin-8 (IL-8) is a potent inflammatory mediator that belongs to the family of C-X-C chemokines. IL-8 promotes the activation and the extravasation of circulating neutrophils to the site of inflammation. Two IL-8 receptor isotypes (type A and B) are identified in human and rabbit neutrophils. IL-8 receptors belongs to the superfamily of G-protein-coupled receptors. Both receptor subtypes A and B bind with high affinity to human IL-8, but they exhibit distinct binding affinity to two functional and structurally related IL-8 peptides, melanoma growth-stimulating activity peptide (MGSA) and neutrophil-activating peptide-2 (NAP-2). Human IL-8 receptor A binds with low affinity to MGSA or NAP-2. In contrast, human IL-8 receptor B binds MGSA with high affinity, and NAP-2 with lesser affinity. Using receptor subtype chimeras, we determined that the N-terminal domain of the receptor confers ligand binding specificity (LaRosa, G. J., Thomas, K. M., Kaufmann, M. E., Mark, R., White, M., Taylor, L., Gray, G., Witt, D., and Navarro, J. (1992) J. Biol. Chem. 267, 25402-25406). In this work, we characterized by molecular cloning and expression a mouse receptor structurally homologous to the IL-8 receptor. We isolated a clone by screening a mouse genomic library with a rabbit IL-8 receptor A cDNA fragment as a probe. The mouse clone exhibited an open reading frame encoding a 359-amino acid protein. Hydropathy plot analysis of the amino acid sequence reveals seven transmembrane domains characteristic of G-protein-coupled receptors. The N terminus and the second extracellular loop contain one and two putative N-glycosylation sites, respectively. The intracellular C-terminal tail contains Ser and Thr residues as potential phosphorylation sites. Northern blot analysis showed that the mouse receptor gene is expressed in mouse neutrophils. The mouse receptor shows 65, 74, 66, and 70% amino acid identity to the rabbit IL-8 receptor subtypes A and B and human IL-8 receptor subtypes A and B, respectively. However, neither mouse neutrophils nor CHO cells expressing the mouse receptor bind human IL-8 in the nanomolar range. To identify the domain(s) conferring high affinity binding to IL-8, we constructed rabbit/mouse receptor chimeras.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Interleucina-8/metabolismo , Estructura Secundaria de Proteína , Receptores de Interleucina/química , Receptores de Interleucina/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Northern Blotting , Células CHO , Cricetinae , Cartilla de ADN , Biblioteca Genómica , Humanos , Cinética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Neutrófilos/inmunología , Neutrófilos/metabolismo , Reacción en Cadena de la Polimerasa , ARN/análisis , Conejos , Receptores de Interleucina/biosíntesis , Receptores de Interleucina-8A , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , TransfecciónRESUMEN
Interleukin-8 (IL-8) mediates the transendothelial migration and activation of neutrophils to the site of inflammation. Two human IL-8 receptor isotype (A and B) and one rabbit IL-8 receptor isotype (A) cDNAs have been previously cloned and characterized on the basis of their pharmacological profile. Human and rabbit IL-8 receptor subtype A binds IL-8 and structurally related peptide melanoma growth-stimulating activity (MGSA) and neutrophil-activating peptide-2 (NAP-2) according to the following affinity binding profile: IL-8 >>> MGSA > NAP-2, whereas the human IL-8 receptor subtype B profile is IL-8 = MGSA > NAP-2 (LaRosa, G., Thomas, K. M., Kaufmann, M., Mark, R., White, M., Taylor, L., Gray, G., Witt, D., and Navarro, J. (1992) J. Biol. Chem. 267, 25402-25406). In this study, we isolated a cDNA clone (5B1a) from a rabbit neutrophil library encoding a G-protein-coupled receptor of the interleukin-8 receptor family. The 5B1a clone encodes a 358-amino acid protein exhibiting 80% amino acid identity to the human IL-8 receptor B, 74% to the rabbit IL-8 receptor A, and 73% to the human IL-8 receptor A. Tissue distribution by Northern blot analysis reveals that the 5B1a mRNA is expressed preferentially in neutrophils. In contrast to previously described IL-8 receptors, the 5B1a receptor exhibited specific 125I-IL-8 binding with a novel affinity binding profile of IL-8 >> NAP-2 > MGSA. The corresponding apparent Ki values for IL-8, NAP-2, and MGSA were 4, 120, and 320 nM, respectively. IL-8 induced intracellular calcium mobilization and desensitization in Chinese hamster ovary cells stably transfected with 5B1a, indicating that this cDNA encodes a functional IL-8 receptor. Sequence analysis of the 5B1a protein with other IL-8 receptor subtypes within the framework of their pharmacological profile reveals putative structural motifs that may correspond to the ligand binding site of the IL-8 receptor.