Functional overexpression and characterization of human bradykinin subtype 2 receptor in insect cells using the baculovirus system

Bradykinin exerts its actions via binding to B1 and B2 receptors (B1R and B2R), which are members of G protein-coupled receptor superfamily. B2R is constitutively expressed in a variety of cells such as endothelial cells, vascular smooth muscle cells, and cardiomyocytes and it is an important drug target for the treatment of cardiovascular disorders. During this study, the human B2R was functionally overexpressed in insect cells using the baculovirus expression system. The maximum expression level in Sf9 cells under optimized condition was 10 pmol/mg. This corresponds to approximately 0.25 mg active receptor per liter culture. The recombinant receptor showed high affinity for its endogenous ligand bradykinin, similar to the B2R expressed in native tissues. Functional coupling of the recombinant receptor to the endogenous G alpha(s) protein was demonstrated via cAMP release assay upon agonist stimulation. Confocal laser scanning microscopy and immunogold-labeling experiment revealed that the recombinant B2R was mainly localized intracellularly and only a minor fraction of the recombinant receptor reached the plasma membrane. To our knowledge, this is the first report of high level expression of recombinant B2R in insect cells and provides a way for large scale production and structural characterization of this receptor.     

Heterologous expression and comparative characterization of the human neuromedin U subtype II receptor using the methylotrophic yeast Pichia pastoris and mammalian cells

Neuromedin U (a neuropeptide) plays regulatory roles in feeding, anxiety, smooth muscle contraction, blood flow and pain. The physiological actions of NmU are mediated via two recently identified G protein-coupled receptors namely the neuromedin U type 1 receptor (NmU(1)R) and the neuromedin U type 2 receptor (NmU(2)R). Despite their crucial roles in cell physiology, structural information on these receptors is limited, mainly due to their low expression levels in native tissues. Here, we report the overexpression of the human NmU(2)R in the methylotrophic yeast Pichia pastoris and baby hamster kidney (BHK) cells using the Semliki Forest virus (SFV) system. The recombinant receptor was expressed as a fusion protein with three different affinity tags namely, the Flag tag, the histidine 10 tag and the biotinylation domain of Propionobacterium shermanii. Expression level of the recombinant receptor was 6-9pmol/mg under optimized conditions, which is significantly higher than the expression level in the native tissues. The recombinant receptor binds to its endogenous ligand neuromedin U with high affinity (Kd=0.8-1.0nM) and the binding constant for the recombinant receptor is similar to that of the wild type NmU(2)R. Enzymatic deglycosylation suggested that the recombinant NmU(2)R was glycosylated in P. pastoris, but not in BHK cells. Confocal laser scanning microscopy and immunogold labelling experiment revealed that the recombinant receptor was predominantly localized in the intracellular membranes. To our knowledge, this is the first report of heterologous overexpression of an affinity tagged recombinant NmU(2)R and it should facilitate further characterization of this receptor.     

Regulation of the human bradykinin B2 receptor expressed in sf21 insect cells: a possible role for tyrosine kinases

The functional regulation of the human bradykinin B2 receptor expressed in sf21 cells was studied. Human bradykinin B2 receptors were immunodetected as a band of 75-80 kDa in membranes from recombinant baculovirus-infected cells and visualized at the plasma membrane, by confocal microscopy, using an antibody against an epitope from its second extracellular loop. B2 receptors, detected in membranes by [(3)H-bradykinin] binding, showed a Kd of 0.66 nmol/L and an expression level of 2.57 pmol/mg of protein at 54 h postinfection. In these cells, bradykinin induced a transient increase of intracellular calcium ([Ca(2+)](i)) in fura 2-AM loaded sf21 cells, and promoted [(35)S]-GTP(gamma)S binding to membranes. The effects of bradykinin were dose dependent (with an EC(50) of 50 nmol/L for calcium mobilization) and were inhibited by N-alpha-adamantaneacetyl-D-Arg-[Hyp(3),Thi(5,8),D-phe(7)]-Bk, a specific B2 receptor antagonist. When the B2 antagonist was applied at the top of the calcium transient, it accelerated the decline of the peak, suggesting that calcium mobilization at this point was still influenced by receptor occupation. No calcium mobilization was elicited by 1 micromol/L (Des-Arg(9))-Bk, a B1 receptor agonist that did not inhibit the subsequent action of 100 nmol/L bradykinin. No effect of bradykinin was detected in uninfected cells or cells infected with the wild-type baculovirus. Bradykinin-induced [Ca(2+)](i) mobilization was increased by genistein and tyrphostin A51. These tyrosine kinase inhibitors did not modify basal levels of [Ca(2+)](i). Homologous desensitization of the B2 receptor was observed after repeated applications of bradykinin, which resulted in attenuated changes in intracellular calcium. In addition, genistein promoted an increased response to a third exposure to the agonist when applied after washing the cells that had been previously challenged with two increasing doses of bradykinin. Genistein did not affect the calcium mobilization induced by activation of the endogenous octopamine G protein-coupled receptor or by thapsigargin. The B2 receptor, detected by confocal microscopy in unpermeabilized cells, remained constant at the surface of cells stimulated with bradykinin for 10 min, in the presence or absence of genistein. Agonist-promoted phosphorylation of the B2 receptor was markedly accentuated by genistein treatment. Phosphoaminoacid analysis revealed the presence of phosphoserine and traces of phosphothreonine, but not phosphotyrosine, suggesting that the putative tyrosine kinase(s), activated by bradykinin, could act in a step previous to receptor phosphorylation. Interestingly, genistein prevented agonist-induced G protein uncoupling from B2 receptors, determined by in vitro bradykinin-stimulated [(35)S]-GTP(gamma)S binding, in membranes from bradykinin pretreated cells. Our results suggest that tyrosine kinase(s) regulate the activity of the human B2 receptor in sf21 cells by affecting its coupling to G proteins and its phosphorylation.     

Dimethylsulphoxide as a tool to increase functional expression of heterologously produced GPCRs in mammalian cells

High-level overexpression of G protein-coupled receptors GPCRs in mammalian cells remains a difficult task inspite of newly developed virus based expression systems. Here, we show that the functional expression level of the recombinant bradykinin receptor (B(2)R) in mammalian cells can be increased up to sixfold just by the addition of dimethylsulphoxide in the culture medium. Total expression level, cellular localization and binding affinity of the recombinant receptor for its endogenous ligand remains unaltered. The strategy presented here, with recombinant B(2)R as a case example, is applicable to other GPCRs and provides a generic tool to improve the functional expression level of recombinant GPCRs in mammalian cells.     

Expression of the alpha 1b-adrenergic receptor and G protein subunits in mammalian cell lines using the Semliki Forest virus expression system

Semliki Forest virus (SFV) vectors have been efficiently used for rapid high level expression of several G protein-coupled receptors. Here we describe the use of SFV vectors to express the alpha 1b-adrenergic receptor (AR) alone or in the presence of the G protein alpha q and/or beta 2 and gamma 2 subunits. Infection of baby hamster kidney (BHK) cells with recombinant SFV-alpha 1b-AR particles resulted in high specific binding activity of the alpha 1b-AR (24 pmol receptor/mg protein). Time-course studies indicated that the highest level of receptor expression was obtained 30 hours post-infection. The stimulation of BHK cells, with epinephrine led to a 5-fold increase in inositol phosphate (IP) accumulation, confirming the functional coupling of the receptor to G protein-mediated activation of phospholipase C. The SFV expression system represents a rapid and reproducible system to study the pharmacological properties and interactions of G protein coupled receptors and of G protein subunits.     

Expression and localization of recombinant human B2 receptors in the methylotrophic yeast Pichia pastoris

The human bradykinin B2 receptor (B2R) fused with green fluorescent protein (GFP) at the C-terminal has been expressed in the methylotrophic yeast of Pichia pastoris. In the expression vector, B2R gene was drove under the highly inducible promoter of alcohol oxidase 1 gene of P. pastoris. By fluorescence activated cell sorting (FACS) analysis and western blot analysis, it was proved that B2R recombinant receptor proteins were expressed at high level in the yeast. Further more, the transformants of P. pastoris were monitored with confocal microscopy, a strong green fluorescence was checked out. The recombinant B2R receptor proteins were mainly located on the plasma membrane proved by immunofluorescence microscopy.     

The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately connected and some of the cardioprotective effects of Losartan are abolished by blocking the bradykinin B2 receptor (B2R) signaling. In this study, we investigated the ability of six clinically available ARBs to specifically bind and activate the B2R. First, we investigated their ability to activate phosphoinositide (PI) hydrolysis in COS-7 cells transiently expressing the B2R. We found that only Losartan activated the B2R, working as a partial agonist compared to the endogenous ligand bradykinin. This effect was blocked by the B2R antagonist HOE 140. A competitive binding analysis revealed that Losartan does not significantly compete with bradykinin and does not change the binding affinity of bradykinin on the B2R. Furthermore, Losartan but not Candesartan mimicked the ability of bradykinin to increase the recovery of contractile force after metabolic stress in rat atrial tissue strips. In conclusion, Losartan is a partial agonist of the B2R through direct binding and activation, suggesting that B2R agonism could partly explain the beneficial effects of Losartan.     

Functional expression and direct visualization of the human alpha 2B -adrenergic receptor and alpha 2B -AR-green fluorescent fusion protein in mammalian cell using Semliki Forest virus vectors

The alpha 2B -adrenergic receptor ( alpha 2B -AR), a member of the G protein-coupled receptor (GPCR) superfamily, was expressed at high levels from Semliki Forest virus (SFV) vectors in mammalian cells. Constructs were engineered by fusing enhanced green fluorescent protein (eGFP) and the SFV capsid to opposite ends of the alpha 2B -AR. The receptor fusions alpha 2B -AR-eGFP and CAP- alpha 2B -AR expressed in CHO-K1 cells generated alpha 2B values of 176 and 122pmol/mg of membrane protein, respectively, and showed similar ligand binding characteristics, alpha 2B -AR subtype-selectivity, and G protein activation as reported for stable expression in CHO-K1 cells. Cryo-electron microscopy and eGFP-based fluorescence indicated the same subcellular receptor distribution. SFV expression is well suited for studies on the pharmacology, biochemistry, and cell biology of GPCRs, and for large-scale recombinant protein production in mammalian suspension culture to generate sufficient receptor quantities for structural biology.     

Localization of bradykinin B(2) receptor in the follicles of porcine ovary and increased expression of matrix metalloproteinase-3 and -20 in cultured granulosa cells by bradykinin treatment.

We have recently shown that not only bradykinin, but also all components for the production of bradykinin, can be detected within the follicle of porcine ovaries. To elucidate the relevance of the intrafollicular bradykinin-producing system to its physiological role, we investigated the distribution of bradykinin receptor (B(2)R) mRNA and the protein in porcine ovaries. A cDNA encoding porcine B(2)R was first cloned from a porcine uterus cDNA library. The receptor mRNA was scarcely detected in the ovary by Northern blot analysis. Polymerase chain reaction analysis with total RNAs isolated from the ovary and from granulosa cells of small and large follicles demonstrated the ovarian expression of B(2)R mRNA. The B(2)R protein was detected by Western blot analysis in extracts of isolated granulosa cells. In situ hybridization of B(2)R mRNA and immunohistochemical analysis of the protein revealed that the receptor is expressed in the theca and granulosa cells of all growing follicles. The effect of bradykinin on the expression of some matrix metalloproteinase (MMP) genes was examined using isolated granulosa cells. Bradykinin treatment induced MMP-3 and MMP-20 gene expression to an extreme degree. The expression of MT1-MMP was also affected by bradykinin treatment. These results suggest that MMPs play a role in follicle rupture during ovulation. The present study provides new information regarding the mechanisms of bradykinin-induced ovulation in porcine ovaries.     

Lack of direct interaction between enalaprilat and the kinin B1 receptors

It has been recently proposed that the second extracellular loop of the human bradykinin (BK) B1 receptor (B1R) contains a conserved HExxH motif also present in peptidases possessing a Zn2+ prosthetic group, such as angiotensin converting enzyme (ACE), and that ACE inhibitors directly activate B1R signaling in endothelial cells. However, the binding of ACE inhibitors to the B1Rs has never been directly evaluated. Information about binding of a radiolabeled inhibitor to natural or recombinant ACE in intact cells (physiologic ionic composition) was also collected. We used the tritiated form of an ACE inhibitor previously proposed to activate the B1R, enalaprilat, to address these questions using recombinant human B1Rs and naturally expressed or recombinant ACE. [3H]Lys-des-Arg9-BK bound to the human recombinant B1Rs with high affinity (KD 0.35 nM) in HEK 293a cells. [3H]Enalaprilat (0.25-10 nM) did not bind to cells expressing recombinant human B1R, but bound with a subnanomolar affinity to recombinant ACE or to naturally expressed ACE in human umbilical vein endothelial cells. The radioligand was further validated using a binding competition assay that involved unlabeled ACE inhibitors or their prodrug forms in endothelial cells. Membranes of HEK 293a cells that expressed B1Rs did not hydrolyze hippuryl-glycylglycine (an ACE substrate). Enalaprilat did not stimulate calcium signaling in HEK 293a cells that expressed B1Rs. A typical ACE inhibitor did not bind to nor stimulate the human B1Rs; nevertheless, several other indirect mechanisms could connect ACE inhibition to B1R stimulation in vivo.     

Roles for heterodimerization of APJ and B2R in promoting cell proliferation via ERK1/2-eNOS signaling pathway

Apelin receptor (APJ) and bradykinin B2 receptor (B2R) play an important role in many physiological processes and share multiple similar characteristics in distribution and functions in the cardiovascular system. We first identified the endogenous expression of APJ and B2R in human umbilical vein endothelial cells (HUVECs) and their co-localization on human embryonic kidney (HEK) 293 cells membrane. A suite of bioluminescence and fluorescence resonance energy transfer (BRET and FRET), proximity ligation assay (PLA), and co-immunoprecipitation (Co-IP) was exploited to demonstrate formation of functional APJ and B2R heterodimer in HUVECs and transfected cells. Stimulation with apelin-13 and bradykinin (BK) increased the phosphorylation of the endothelial nitric oxide synthase (eNOS) in HUVECs, which could be inhibited by the silencing of APJ or B2R, indicating the APJ-B2R dimer is critical for eNOS phosphorylation in HUVECs. Furthermore, the increase of NOS and extracellular signal regulated kinases1/2 (ERK1/2) phosphorylation mediated by APJ/B2R dimer can be inhibited by U0126 and U73122, respectively, suggesting that the heterodimer might activate the PLC/ERK1/2/eNOS signaling pathway, and finally leading to a significant increase in cell proliferation. Thus, we uncovered for the first time the existence of APJ-B2R heterodimer and provided a promising new target in cardiovascular therapeutics.     

A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor

Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1–EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor’s amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of co-enriched receptor proteins immobilized on agarose beads also detected a high FRET efficiency of 24.0%. Taken together confocal FRET imaging revealed efficient heterodimerization of co-enriched and cellular AT1/B2R, and GRK-dependent co-internalization of the AT1/B2R heterodimer.     

Cross-talk between carboxypeptidase M and the kinin B1 receptor mediates a new mode of G protein-coupled receptor signaling

G protein-coupled receptor (GPCR) signaling is affected by formation of GPCR homo- or heterodimers, but GPCR regulation by other cell surface proteins is not well understood. We reported that the kinin B1 receptor (B1R) heterodimerizes with membrane carboxypeptidase M (CPM), facilitating receptor signaling via CPM-mediated conversion of bradykinin or kallidin to des-Arg kinin B1R agonists. Here, we found that a catalytically inactive CPM mutant that still binds substrate (CPM-E264Q) also facilitates efficient B1R signaling by B2 receptor agonists bradykinin or kallidin. This response required co-expression of B1R and CPM-E264Q in the same cell, was disrupted by antibody that dissociates CPM from B1R, and was not found with a CPM-E264Q-B1R fusion protein. An additional mutation that reduced the affinity of CPM for C-terminal Arg and increased the affinity for C-terminal Lys inhibited the B1R response to bradykinin (with C-terminal Arg) but generated a response to Lys(9)-bradykinin. CPM-E264Q-mediated activation of B1Rs by bradykinin resulted in increased intramolecular fluorescence resonance energy transfer (FRET) in a B1R FRET construct, similar to that generated directly by a B1R agonist. In cytokine-treated human lung microvascular endothelial cells, disruption of B1R-CPM heterodimers inhibited B1R-dependent NO production stimulated by bradykinin and blocked the increased endothelial permeability caused by treatment with bradykinin and pyrogallol (a superoxide generator). Thus, CPM and B1Rs on cell membranes form a critical complex that potentiates B1R signaling. Kinin peptide binding to CPM causes a conformational change in the B1R leading to intracellular signaling and reveals a new mode of GPCR activation by a cell surface peptidase.     

Desensitization of GABA(B) receptor signaling by formation of protein complexes of GABA(B2) subunit with GRK4 or GRK5

We investigated the role of G protein coupled-receptor kinases (GRKs) in the desensitization of GABA(B) receptor-mediated signaling using Xenopus oocytes and baby hamster kidney (BHK) cells. Baclofen elicited inward K(+) currents in oocytes coexpressing heterodimeric GABA(B) receptor, GABA(B1a) subunit (GB(1a)R) and GABA(B2) subunit (GB(2)R), together with G protein-activated inwardly rectifying K(+) channels (GIRKs), in a concentration-dependent manner. Repetitive application of baclofen to oocytes coexpressing GABA(B)R and GIRKs did not change peak K(+) currents in the first and second responses, but the latter responses were significantly attenuated by coexpression of either GRK4 or GRK5 with attenuation efficacy of GRK4 > GRK5. Coexpression of other GRKs including GRK2, GRK3, and GRK6 had no effect on GABA(B) receptor-mediated desensitization processes. In BHK cells coexpressing GRK4 fused to Venus (brighter variant of yellow fluorescent protein, GRK4-Venus) with GB(1a)R and GB(2)R, GRK4-Venus was expressed in the cytosol but was translocated to the plasma membranes by GABA(B)R activation. In BHK cells coexpressing GRK4 fused to Cerulean (brighter variant of cyan fluorescent protein, GRK4-Cerulean) with GB(1a)R and GB(2)R-Venus, fluorescence resonance energy transfer (FRET) analysis demonstrated that GRK4-Cerulean formed a protein complex with GB(2)R-Venus. Immunoprecipitation and Western blot analysis confirmed GB(2)R-GRK4 complex formation. GRK5 also formed a complex with GB(2)R on the plasma membranes as determined by FRET and Western blotting but not GRK2, GRK3, and GRK6. Our results indicate that GRK4 and GRK5 desensitize GABA(B) receptor-mediated responses by forming protein complexes with GB(2)R subunit of GABA(B)R at the plasma membranes.     

Regulation of kinin B(2) receptors by bradykinin in human lung cells

Bradykinin is a potent mediator of inflammation that has been shown to participate in allergic airway inflammation. The biologic effects of bradykinin are mediated by binding and activation of its cognate receptor, the B(2) receptor (B(2)R). In the lung fibroblast cell line IMR-90, binding of bradykinin to B(2)R triggers down-regulation of receptor surface expression, suggesting that bradykinin-induced inflammation is transient and self-limited. Notably, subjects with chronic airway inflammation continue to respond to BK following a first challenge. B(2)Rs are expressed on many different lung cell types, including airway epithelial cells. We therefore compared IMR-90 cells with the human lung epithelial cell line BEAS2B and found that B(2)R expression in the two cell types is differently regulated by BK. Whereas BK induces down-regulation of B(2)R in IMR-90 cells, the same treatment leads to up-regulation of the receptor in BEAS2B cells. These results provide a possible explanation for the potency of bradykinin in inducing ongoing airway inflammation.     

Spontaneous formation of a proteolytic B1 and B2 bradykinin receptor complex with enhanced signaling capacity

B1 bradykinin receptor (B1R) induction is critical in the adaptation of the kinin-mediated inflammatory response from a B2 bradykinin receptor (B2R) subtype to a B1R subtype that occurs during chronic insult. Here, we show that B1R spontaneously forms a proteolytic plasma membrane complex with B2R along with increased receptor signaling capacity. Co-expression of hemagglutinin-tagged B2R with FLAG-tagged B1R in HEK293 cells resulted in degradation of B2R as determined by the diminution of the intact 65-kDa B2R species and the appearance of proteolytic B2R products at 30-40 kDa and by the reduction in B2R bradykinin binding sites. On the other hand, the 35-kDa B1R remained intact. Receptor co-expression also led to an increase in constitutive and agonist-stimulated receptor signaling. Selective immunoprecipitation with epitope-specific antibodies revealed a spontaneously formed heterologous receptor complex, which was composed of the intact 35-kDa B1R and the B2R degradation products. Cellular fractionation, cell surface biotinylation, and immunoelectron microscopy showed that B2R.B1R complexes were present on the cell surface. This is the first evidence that a heterologous G protein-coupled receptor complex in the plasma membrane is linked to proteolytic degradation of a participating receptor, and this mechanism may contribute to the adaptation of the kinin response from a B2 type to a B1 type during chronic insult.     

人GLP-1R基因转染BHK细胞及重组细胞的应用

为了构建表达人胰高血糖素样肽-1受体(GLP-1R)基因的BHK细胞株,并利用该重组细胞对GLP-1等相关肽进行活性测定,首先通过酶切、连接方式将人GLP-1R基因克隆至真核表达载体pCDNA3.(1 )中,然后用脂质体转染法将重组质粒转染至BHK-21细胞,转染后的细胞经G418加压筛选、细胞有限稀释等方法获得克隆细胞株。经过该细胞株RT-PCR验证,结果证实目的基因已整合至BHK-21细胞基因组中,并获得成功转录和表达。活性检测实验表明该重组细胞株经过GLP-1的刺激后,其细胞中的cAMP含量得到明显提升。该细胞株的构建为GLP-1及相关肽的活性测定奠定了基础。     

Fever-like temperature modification differentially affects in vitro signaling of bradykinin B(1) and B(2) receptors

The bradykinin (BK) B(2) and B(1) receptors (B(2)R, B(1)R) belong to the rhodopsin-like G protein-coupled receptors (GPCRs) and are involved in (patho)physiological processes such as blood pressure regulation or inflammation. They mediate the effects of the pro-inflammatory peptides bradykinin/kallidin and desArg(9)-BK/desArg(10)-kallidin, respectively. Whereas the B(2)R is constitutively expressed and gets internalized upon activation, the B(1)R is especially induced by inflammatory mediators and responds to stimulation with increased surface receptor numbers. Stimulation of both receptors activates phospholipase Cβ (PLCβ) and mitogen activated protein kinase (MAPK) signaling. Because inflammatory processes are characterized by heat (fever), we analyzed the effect of increased temperature (41°C vs. 37°C) on B(1)R and B(2)R signaling in HEK 293 and IMR 90 cells. Our results show that signaling of both receptors is temperature-sensitive, however to a different extent and with regard to the investigated pathways. Comparing PLCβ activity and Ca(2+)-regulated signals, a temperature-dependent increase was only observed for B(1)R but not for B(2)R activation, whereas MAPK activities were doubled at 41°C for both receptors. Taken together, our findings suggest that the observed temperature sensitivity of B(1)R-induced PLCβ activation is B(1)R-specific. In contrast, the enhanced stimulation of MAPK activity under hyperthermic conditions appears to be a common phenomenon for GPCRs.     

Immunolocalization and expression of kinin B1R and B2R receptors in human inflammatory bowel disease

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B(1)R) and bradykinin receptor 2 (B(2)R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B(2)R and B(1)R proteins are expressed in the epithelial cells of normal and IBD intestines. B(1)R protein is visualized in macrophages at the center of granulomas in CD. B(2)R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B(1)R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B(1)R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B(1)R mRNA is significantly higher than B(2)R mRNA. However, in inactive UC patients, the B(1)R and B(2)R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B(1)R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.     

K317, R319, and E320 within the proximal C-terminus of the bradykinin B2 receptor form a motif important for phospholipase C and phospholipase A2 but not connective tissue growth factor related signaling

We showed previously that large domain exchanges between the bradykinin B2 (BKB2) and angiotensin II type 1a (AT1a) receptors can result in functional hybrids. However, when we proceeded to exchange the entire bradykinin B2 receptor (BKB2R) C-terminal tail with the AT1aR C-terminus, the hybrid, while continuing to bind BK and be endocytosed as wild type (WT) BKB2R, lost much of its ability to activate phosphatidylinositol (PI) turnover or the release of arachidonic acid (ARA). In this study, we constructed chimeric receptors within the proximal C-terminus between the BKB2R and AT1aR or bradykinin B1 receptor (BKB1R). The mutant and WT receptor cDNAs were stably transfected into Rat-1 cells. Also, point mutations were generated to evaluate the role of the individual residues within this region. These chimeric studies revealed that the proximal portion of the BKB2R C-tail is crucial for G protein-linked BKB2R functions. This region could not be swapped with the AT1aR to obtain a BK activated PI turnover or ARA release. Further studies demonstrated that the distal portion (325-330) of this region is exchangeable; however, the middle portion (317-324) is not. Small motif exchanges within this section identified the KSR and EVY motifs as crucial for G(alphaq), G(alphai) related signaling of the BKB2R. Point mutations then showed that the charged amino acids K317, R319, and E320 are the residues critical for linking to PI turnover and ARA release. However, these proximal chimeras showed normal receptor uptake. Interestingly, while apparently not activating G protein-linked signaling, the proximal tail AT1aR exchange mutant and the entire C-terminus exchange hybrid continued to cause a substantial bradykinin effected increase in connective tissue growth factor (CTGF) mRNA level, as WT BKB2R.