Downregulation of kinin B1 receptor function by B2 receptor heterodimerization and signaling.

Signaling through the G protein-coupled kinin receptors B1 (kB1R) and B2 (kB2R) plays a critical role in inflammatory responses mediated by activation of the kallikrein-kinin system. The kB2R is constitutively expressed and rapidly desensitized in response to agonist whereas kB1R expression is upregulated by inflammatory stimuli and it is resistant to internalization and desensitization. Here we show that the kB1R heterodimerizes with kB2Rs in co-transfected HEK293 cells and natively expressing endothelial cells, resulting in significant internalization and desensitization of the kB1R response in cells pre-treated with kB2R agonist. However, pre-treatment of cells with kB1R agonist did not affect subsequent kB2R responses. Agonists of other G protein-coupled receptors (thrombin, lysophosphatidic acid) had no effect on a subsequent kB1R response. The loss of kB1R response after pretreatment with kB2R agonist was partially reversed with kB2R mutant Y129S, which blocks kB2R signaling without affecting endocytosis, or T342A, which signals like wild type but is not endocytosed. Co-endocytosis of the kB1R with kB2R was dependent on ß-arrestin and clathrin-coated pits but not caveolae. The sorting pathway of kB1R and kB2R after endocytosis differed as recycling of kB1R to the cell surface was much slower than that of kB2R. In cytokine-treated human lung microvascular endothelial cells, pre-treatment with kB2R agonist inhibited kB1R-mediated increase in transendothelial electrical resistance (TER) caused by kB1R stimulation (to generate nitric oxide) and blocked the profound drop in TER caused by kB1R activation in the presence of pyrogallol (a superoxide generator). Thus, kB1R function can be downregulated by kB2R co-endocytosis and signaling, suggesting new approaches to control kB1R signaling in pathological conditions.

Carboxypeptidase M is a positive allosteric modulator of the kinin B1 receptor.

Ligand binding to extracellular domains of G protein-coupled receptors can result in novel and nuanced allosteric effects on receptor signaling. We previously showed that the protein-protein interaction of carboxypeptidase M (CPM) and kinin B1 receptor (B1R) enhances B1R signaling in two ways; 1) kinin binding to CPM causes a conformational activation of the B1R, and 2) CPM-generated des-Arg-kinin agonist is efficiently delivered to the B1R. Here, we show CPM is also a positive allosteric modulator of B1R signaling to its agonist, des-Arg(10)-kallidin (DAKD). In HEK cells stably transfected with B1R, co-expression of CPM enhanced DAKD-stimulated increases in intracellular Ca(2+) or phosphoinositide turnover by a leftward shift of the dose-response curve without changing the maximum. CPM increased B1R affinity for DAKD by ∼5-fold but had no effect on basal B1R-dependent phosphoinositide turnover. Soluble, recombinant CPM bound to HEK cells expressing B1Rs without stimulating receptor signaling. CPM positive allosteric action was independent of enzyme activity but depended on interaction of its C-terminal domain with the B1R extracellular loop 2. Disruption of the CPM/B1R interaction or knockdown of CPM in cytokine-treated primary human endothelial cells inhibited the allosteric enhancement of CPM on B1R DAKD binding or ERK1/2 activation. CPM also enhanced the DAKD-induced B1R conformational change as detected by increased intramolecular fluorescence or bioluminescence resonance energy transfer. Thus, CPM binding to extracellular loop 2 of the B1R results in positive allosteric modulation of B1R signaling, and disruption of this interaction could provide a novel therapeutic approach to reduce pathological B1R signaling.

Carboxypeptidase M augments kinin B1 receptor signaling by conformational crosstalk and enhances endothelial nitric oxide output.

The G protein-coupled receptors (GPCRs) are the largest class of membrane proteins that play key roles in transducing extracellular signals to intracellular proteins to generate cellular responses. The kinin GPCRs, named B1 (B1R) and B2 (B2R), are responsible for mediating the biological responses to kinin peptides released from the precursor kininogens. Bradykinin (BK) or kallidin (KD) are agonists for B2Rs, whereas their carboxypeptidase (CP)-generated metabolites, des-Arg(9)-BK or des-Arg(10)-KD, are specific agonists for B1Rs. Here, we review the evidence for a critical role of membrane-bound CPM in facilitating B1R signaling by its ability to directly activate the receptor via conformational crosstalk as well as generate its specific agonist. In endothelial cells, the CPM/B1R interaction facilitates B1R-dependent high-output nitric oxide under inflammatory conditions.

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.

Angiotensin I-converting enzyme inhibitors are allosteric enhancers of kinin B1 and B2 receptor function.

The beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors go beyond the inhibition of ACE to decrease angiotensin (Ang) II or increase kinin levels. ACE inhibitors also affect kinin B1 and B2 receptor (B1R and B2R) signaling, which may underlie some of their therapeutic usefulness. They can indirectly potentiate the actions of bradykinin (BK) and ACE-resistant BK analogs on B2Rs to elevate arachidonic acid and NO release in laboratory experiments. Studies indicate that ACE inhibitors and some Ang metabolites increase B2R functions as allosteric enhancers by inducing a conformational change in ACE. This is transmitted to B2Rs via heterodimerization with ACE on the plasma membrane of cells. ACE inhibitors are also agonists of the B1R, at a Zn-binding sequence on the second extracellular loop that differs from the orthosteric binding site of the des-Arg-kinin peptide ligands. Thus, ACE inhibitors act as direct allosteric B1R agonists. When ACE inhibitors enhance B2R and B1R signaling, they augment NO production. Enhancement of B2R signaling activates endothelial NO synthase, yielding a short burst of NO; activation of B1Rs results in a prolonged high output of NO by inducible NO synthase. These actions, outside inhibiting peptide hydrolysis, may contribute to the pleiotropic therapeutic effects of ACE inhibitors in various cardiovascular disorders.

Estrogen replacement therapy in diabetic ovariectomized female rats potentiates postischemic leukocyte adhesion in cerebral venules via a RAGE-related process.

In this study, we tested the hypothesis that the documented transformation of 17beta-estradiol (E2) from a counterinflammatory hormone in nondiabetic (ND) rats to a proinflammatory agent in rats with diabetes mellitus (DM) is due to an enhanced contribution from the receptor for advanced glycation end products (RAGE). Rhodamine 6G-labeled leukocytes were observed through a closed cranial window in rats. In vivo pial venular leukocyte adherence and infiltration were measured over 10 h reperfusion after transient forebrain ischemia in DM (streptozotocin) versus ND intact, ovariectomized (OVX), and E2-replaced (for 7-10 days) OVX (OVE) females. The role of RAGE was examined in two ways: 1) RAGE knockdown via topical application of RAGE antisense versus missense oligodeoxynucleotide or 2) intracerebroventricular injection of the RAGE decoy inhibitor, soluble RAGE. Among diabetic rats, the lowest levels of cortical RAGE mRNA and immunoreactivity of the RAGE ligand, AGE, were seen in OVX females, with significantly higher levels exhibited in intact and OVE females. However, results from the analysis of cortical RAGE protein only partially tracked those findings. When comparing ND to DM rats, cortical AGE immunoreactivity was significantly lower in OVE and intact females but similar in OVX rats. In DM rats, the level of postischemic leukocyte adhesion and infiltration (highest to lowest) was OVE>intact>>untreated OVX. In NDs, adhesion was highest in the untreated OVX group. Leukocyte extravasation was observed at >6 h postischemia but only in diabetic OVE and intact females and in ND OVX (untreated) rats. Pretreatment with RAGE antisense-oligodeoxynucleotide or soluble RAGE attenuated postischemic leukocyte adhesion and prevented infiltration but only in the diabetic OVE and intact groups. These results indicate that the exacerbation of postischemic leukocyte adhesion by chronic E2 replacement therapy in diabetic OVX females involves a RAGE-related mechanism. Targeting RAGE may restore the neuroprotective effect of E2 replacement therapy in diabetic females.

Carboxypeptidase M and kinin B1 receptors interact to facilitate efficient b1 signaling from B2 agonists.

Kinin B1 receptor (B1R) expression is induced by injury or inflammatory mediators, and its signaling produces both beneficial and deleterious effects. Kinins cleaved from kininogen are agonists of the B2R and must be processed by a carboxypeptidase to generate B1R agonists des-Arg(9)-bradykinin or des-Arg(10)-kallidin. Carboxypeptidase M (CPM) is a membrane protein potentially well suited for this function. Here we show that CPM expression is required to generate a B1R-dependent increase in [Ca(2+)](i) in cells stimulated with B2R agonists kallidin or bradykinin. CPM and the B1R interact on the cell membrane, as shown by co-immunoprecipitation, cross-linking, and fluorescence resonance energy transfer analysis. CPM and B1R are also co-localized in lipid raft/caveolin-enriched membrane fractions, as determined by gradient centrifugation. Treatment of cells co-expressing CPM and B1R with methyl-beta-cyclodextrin to disrupt lipid rafts reduced the B1R-dependent increase in [Ca(2+)](i) in response to B2R agonists, whereas cholesterol treatment enhanced the response. A monoclonal antibody to the C-terminal beta-sheet domain of CPM reduced the B1R response to B2R agonists without inhibiting CPM. Cells expressing a novel fusion protein containing CPM at the N terminus of the B1R also increased [Ca(2+)](i) when stimulated with B2R agonists, but the response was not reduced by methyl-beta-cyclodextrin or CPM antibody. A B1R- and CPM-dependent calcium signal in response to B2R agonist bradykinin was also found in endothelial cells that express both proteins. Thus, a close relationship of B1Rs and CPM on the membrane is required for efficiently generating B1R signals, which play important roles in inflammation.

Dynamic receptor-dependent activation of inducible nitric-oxide synthase by ERK-mediated phosphorylation of Ser745.

Nitric oxide (NO) is a pleiotropic regulator of vascular function, and its overproduction by inducible nitric-oxide synthase (iNOS) in inflammatory conditions plays an important role in the pathogenesis of vascular diseases. iNOS activity is thought to be regulated primarily at the level of expression to generate "high output" NO compared with constitutive NO synthases. Here we show iNOS activity is acutely up-regulated by activation of the B1-kinin receptor (B1R) in human endothelial cells or transfected HEK293 cells to generate 2.5-5-fold higher NO than that stimulated by Arg alone. Increased iNOS activity was dependent on B1R activation of the MAPK ERK. In HEK293 cells transfected with human iNOS and B1R, ERK phosphorylated iNOS on Ser745 as determined by Western analysis using phospho-Ser antibody, in vitro kinase assays with activated ERK, and MALDI-TOF mass spectrometry. Mutation of Ser745 to Ala did not affect basal iNOS activity but eliminated iNOS phosphorylation and activation in response to B1R agonist. Mutation of Ser745 to Asp resulted in a basally hyperactive iNOS whose activity was not further increased by B1R agonist. ERK and phospho-ERK (after B1R activation) were co-localized with iNOS as determined by confocal fluorescence microscopy. Furthermore, ERK co-immunoprecipitated with iNOS. The discovery that iNOS can be phosphorylated by ERK and acutely activated by receptor-mediated signaling reveals a new level of regulation for this isoform. These findings provide a novel therapeutic target to explore in the treatment of vascular inflammatory diseases.

Crystal structure of the human carboxypeptidase N (kininase I) catalytic domain.

Human carboxypeptidase N (CPN), a member of the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, is an extracellular glycoprotein synthesized in the liver and secreted into the blood, where it controls the activity of vasoactive peptide hormones, growth factors and cytokines by specifically removing C-terminal basic residues. Normally, CPN circulates in blood plasma as a hetero-tetramer consisting of two 83 kDa (CPN2) domains each flanked by a 48 to 55 kDa catalytic (CPN1) domain. We have prepared and crystallized the recombinant C-terminally truncated catalytic domain of human CPN1, and have determined and refined its 2.1 A crystal structure. The structural analysis reveals that CPN1 has a pear-like shape, consisting of a 319 residue N-terminal catalytic domain and an abutting, cylindrically shaped 79 residue C-terminal beta-sandwich transthyretin (TT) domain, more resembling CPD-2 than CPM. Like these other CPN/E members, two surface loops surrounding the active-site groove restrict access to the catalytic center, offering an explanation for why some larger protein carboxypeptidase inhibitors do not inhibit CPN. Modeling of the Pro-Phe-Arg C-terminal end of the natural substrate bradykinin into the active site shows that the S1' pocket of CPN1 might better accommodate P1'-Lys than Arg residues, in agreement with CPN's preference for cleaving off C-terminal Lys residues. Three Thr residues at the distal TT edge of CPN1 are O-linked to N-acetyl glucosamine sugars; equivalent sites in the membrane-anchored CPM are occupied by basic residues probably involved in membrane interaction. In tetrameric CPN, each CPN1 subunit might interact with the central leucine-rich repeat tandem of the cognate CPN2 subunit via a unique hydrophobic surface patch wrapping around the catalytic domain-TT interface, exposing the two active centers.

Human ACE and bradykinin B2 receptors form a complex at the plasma membrane.

To investigate how angiotensin I-converting enzyme (ACE) inhibitors enhance the actions of bradykinin (BK) on B2 receptors independent of blocking BK inactivation, we expressed human somatic ACE and B2 receptors in CHO cells. Bradykinin and its ACE-resistant analog were the receptor agonists. B2 fused with green fluorescent protein (GFP) and ACE were coprecipitated with antisera to GFP or ACE shown in Western blots. Immunohistochemistry of fixed cells localized ACE by red color and B2-GFP by green. Yellow on plasma membranes of coexpressing cells also indicated enzyme-receptor complex formation. Using ACE-fused cyan fluorescent protein donor and B2-fused yellow fluorescent protein (YFP) acceptor, we registered fluorescence resonance energy transfer (FRET) by the enhanced fluorescence of donor on acceptor photobleaching, establishing close (within 10 nm) positions of B2 receptors and ACE. Bradykinin stimulation cointernalized ACE and B2 receptors. We expressed ACE fused to N terminus of B2 receptors, anchoring only receptors to plasma membranes. Here, in contrast to cells, where both ACE and B2 receptors are separately anchored, ACE inhibitors neither enhance activation of chimeric B2 nor resensitize desensitized B2 receptors. Heterodimer formation between ACE and B2 receptors can be a mechanism for ACE inhibitors to augment kinin activity at cellular level.

Positive cooperativity between the thrombin and bradykinin B2 receptors enhances arachidonic acid release.

Bradykinin (BK) or kallikreins activate B2 receptors (R) that couple Galpha(i) and Galpha(q) proteins to release arachidonic acid (AA) and elevate intracellular Ca2+ concentration ([Ca2+]i). Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples Galpha(i), Galpha(q), and Galpha(12/13) proteins. In Chinese hamster ovary cells stably transfected with human B2R, thrombin liberated little AA, but it significantly potentiated AA release by B2R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B2R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of Galpha(i), Galpha(q), and Galpha(12/13) signaling pathways, and a protein kinase C (PKC)-alpha inhibitor, Gö-6976, blocked potentiation, while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca2+]i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca2+]i by thrombin and blocked potentiation of AA release by B2R agonists. Because either a nonselective inhibitor (isotetrandrine) of phospholipase A2 (PLA2) or a Ca2+-dependent PLA2 inhibitor abolished potentiation of AA release by thrombin, while a Ca2+-independent PLA2 inhibitor did not, we concluded that the mechanism involves Ca2+-dependent PLA2 activation. Both thrombin and TRAP modified activation and phosphorylation of the B2R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the NH2-terminal Ser1-Phe2 bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus PAR1 activation enhances AA release by B2R agonists through signal transduction pathway.

Kallikrein activates bradykinin B2 receptors in absence of kininogen.

Kallikreins cleave plasma kininogens to release the bioactive peptides bradykinin (BK) or kallidin (Lys-BK). These peptides then activate widely disseminated B2 receptors with consequences that may be either noxious or beneficial. We used cultured cells to show that kallikrein can bypass kinin release to activate BK B2 receptors directly. To exclude intermediate kinin release or kininogen uptake from the cultured medium, we cultured and maintained cells in medium entirely free of animal proteins. We compared the responses of stably transfected Chinese hamster ovary (CHO) cells that express human B2 receptors (CHO B2) and cells that coexpress angiotensin I-converting enzyme (ACE) as well (CHO AB). We found that BK (1 nM or more) and tissue kallikrein (1-10 nM) both significantly increased release of arachidonic acid beyond unstimulated baseline level. An enzyme-linked immunoassay for kinin established that kallikrein did not release a kinin from CHO cells. We confirmed the absence of kininogen mRNA with RT-PCR to rule out kininogen synthesis by CHO cells. We next tested an ACE inhibitor for enhanced BK receptor activation in the absence of kinin release and synthesized an ACE-resistant BK analog as a control for these experiments. Enalaprilat (1 microM) potentiated kallikrein (100 nM) in CHO AB cells but was ineffective in CHO B2 cells that do not bear ACE. We concluded that kallikrein activated B2 receptors without releasing a kinin. Furthermore, inhibition of ACE enhanced the receptor activation by kallikrein, an action that may contribute to the manifold therapeutic effects of ACE inhibitors.

Hydrolysis of angiotensin peptides by human angiotensin I-converting enzyme and the resensitization of B2 kinin receptors.

We measured the cleavage of angiotensin I (Ang I) metabolites by angiotensin I-converting enzyme (ACE) in cultured cells and examined how they augment actions of bradykinin B2 receptor agonists. Monolayers of Chinese hamster ovary cells transfected to stably express human ACE and bradykinin B2 receptors coupled to green fluorescent protein (B2GFP) or to express only coupled B2GFP receptors. We used 2 ACE-resistant bradykinin analogues to activate the B2 receptors. We used high-performance liquid chromatography to analyze the peptides cleaved by ACE on cell monolayers and found that Ang 1-9 was hydrolyzed 18x slower than Ang I and &30% slower than Ang 1-7. Ang 1-7 was cleaved to Ang 1-5. Although micromol/L concentrations of slowly cleaved substrates Ang 1-7 and Ang 1-9 inhibit ACE, they resensitize the desensitized B2GFP receptors in nmol/L concentration, independent of ACE inhibition. This is reflected by release of arachidonic acid through a mechanism involving cross-talk between ACE and B2 receptors. When ACE was not expressed, the Ang 1-9, Ang 1-7 peptides were inactive. Inhibitors of protein kinase C-alpha, phosphatases and Tyr-kinase blocked this resensitization activity, but not basal B2 activation by bradykinin. Ang 1-9 and Ang 1-7 enhance bradykinin activity, probably by acting as endogenous allosteric modifiers of the ACE and B2 receptor complex. Consequently, when ACE inhibitors block conversion of Ang I, other enzymes can still release Ang I metabolites to enhance the efficacy of ACE inhibitors.

Plasmin alters the activity and quaternary structure of human plasma carboxypeptidase N.

Human CPN (carboxypeptidase N) is a tetrameric plasma enzyme containing two glycosylated 83 kDa non-catalytic/regulatory subunits that carry and protect two active catalytic subunits. Because CPN can regulate the level of plasminogen binding to cell surface proteins, we investigated how plasmin cleaves CPN and the consequences. The products of hydrolysis were analysed by activity assays, Western blotting, gel filtration and sequencing. When incubated with intact CPN tetramer, plasmin rapidly cleaved the 83 kDa subunit at the Arg457-Ser458 bond near the C-terminus to produce fragments of 72 and 13 kDa, thereby releasing an active 142 kDa heterodimer, and also cleaved the active subunit, decreasing its size from 55 kDa to 48 kDa. Further evidence for the heterodimeric form of CPN was obtained by re-complexing the non-catalytic 72 kDa fragment with recombinant catalytic subunit or by immunoprecipitation of the catalytic subunit after plasmin treatment of CPN using an antibody specific for the 83 kDa subunit. Upon longer incubation, plasmin cleaved the catalytic subunit at Arg218-Arg219 to generate fragments of 27 kDa and 21 kDa, held together by non-covalent bonds, that were more active than the native enzyme. These data show that plasmin can alter CPN structure and activity, and that the C-terminal 13 kDa fragment of the CPN 83 kDa subunit is a docking peptide that is necessary to maintain the stable active tetrameric form of human CPN in plasma.

Crystal structure of human carboxypeptidase M, a membrane-bound enzyme that regulates peptide hormone activity.

Carboxypeptidase M (CPM), an extracellular glycosylphosphatidyl-inositol(GPI)-anchored membrane glycoprotein belonging to the CPN/E subfamily of "regulatory" metallo-carboxypeptidases, specifically removes C-terminal basic residues from peptides and proteins. Due to its wide distribution in human tissues, CPM is believed to play important roles in the control of peptide hormone and growth factor activity at the cell surface, and in the membrane-localized degradation of extracellular proteins. We have crystallized human GPI-free CPM, and have determined and refined its 3.0A crystal structure. The structure analysis reveals that CPM consists of a 295 residue N-terminal catalytic domain similar to that of duck CPD-2 (but only distantly related to CPA/B), an adjacent 86 residue beta-sandwich C-terminal domain characteristic of the CPN/E family but more conically shaped than the equivalent domain in CPD-2, and a unique, partially disordered 25 residue C-terminal extension to which the GPI membrane-anchor is post-translationally attached. Through this GPI anchor, and presumably via some positively charged side-chains of the C-terminal domain, the CPM molecule may interact with the membrane in such a way that its active centre will face alongside, i.e. well suited to interact with other membrane-bound protein substrates or small peptides. Modelling of the C-terminal part of the natural substrate Arg(6)-Met-enkephalin into the active site shows that the S1' pocket of CPM is particularly well designed to accommodate P1'-Arg residues, in agreement with the preference of CPM for cleaving C-terminal Arg.

The ectodomain shedding of angiotensin-converting enzyme is independent of its localisation in lipid rafts.

Angiotensin-converting enzyme (ACE), a type I integral membrane protein that plays a major role in vasoactive peptide metabolism, is shed from the plasma membrane by proteolytic cleavage within the juxtamembrane stalk. To investigate whether this shedding is regulated by lateral segregation in cholesterol-rich lipid rafts, Chinese hamster ovary cells and human neuroblastoma SH-SY5Y cells were transfected with either wild-type ACE (WT-ACE) or a construct with a glycosylphosphatidylinositol (GPI) anchor attachment signal replacing the transmembrane and cytosolic domains (GPI-ACE). In both cell types, GPI-ACE, but not WT-ACE, was sequestered in caveolin or flotillin-enriched lipid rafts and was released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C. When cells were treated with activators of the protein kinase C signalling cascade (phorbol myristate acetate or carbachol) the shedding of GPI-ACE was stimulated to a similar extent to that of WT-ACE. The release of WT-ACE and GPI-ACE from the cells was inhibited in an identical manner by a range of hydroxamate-based zinc metalloprotease inhibitors. Disruption of lipid rafts by filipin treatment did not alter the shedding of GPI-ACE, and phorbol ester treatment did not alter the distribution of WT-ACE or GPI-ACE between raft and non-raft membrane compartments. These data clearly show that the protein kinase C-stimulated shedding of ACE does not require the transmembrane or cytosolic regions of the protein, and that sequestration in lipid rafts does not regulate the shedding of the protein.

Effect of mutation of two critical glutamic acid residues on the activity and stability of human carboxypeptidase M and characterization of its signal for glycosylphosphatidylinositol anchoring.

Human carboxypeptidase (CP) M was expressed in baculovirus-infected insect cells in a glycosylphosphatidylinositol-anchored form, whereas a truncated form, lacking the putative signal sequence for glycosylphosphatidylinositol anchoring, was secreted at high levels into the medium. Both forms had lower molecular masses (50 kDa) than native placental CPM (62 kDa), indicating minimal glycosylation. The predicted glycosylphosphatidylinositol-anchor attachment site was investigated by mutation of Ser(406) to Ala, Thr or Pro and expression in HEK-293 and COS-7 cells. The wild-type and S406A and S406T mutants were expressed on the plasma membrane in glycosylphosphatidylinositol-anchored form, but the S406P mutant was not and was retained in a perinuclear location. The roles of Glu(260) and Glu(264) in CPM were investigated by site-directed mutagenesis. Mutation of Glu(260) to Gln had minimal effects on kinetic parameters, but decreased heat stability, whereas mutation to Ala reduced the k(cat)/ K(m) by 104-fold and further decreased stability. In contrast, mutation of Glu(264) to Gln resulted in a 10000-fold decrease in activity, but the enzyme still bound to p-aminobenzoylarginine-Sepharose and was resistant to trypsin treatment, indicating that the protein was folded properly. These results show that Glu(264) is the critical catalytic glutamic acid and that Glu(260) probably stabilizes the conformation of the active site.

Products of angiotensin I hydrolysis by human cardiac enzymes potentiate bradykinin.

Some beneficial effects of ACE inhibitors are attributed to potentiation of bradykinin's actions exerted through its B2 receptor. We investigated them on cultured cells transfected or constitutively expressing both ACE and B2 receptor. The potentiation of bradykinin was indirect and attributed to a crosstalk induced between enzyme and receptor via ACE, a heterodimer formation. While looking for endogenous activators, we investigated the split products of angiotensin I (Ang) Ang 1-9 and 1-7, peptides released by enzymes of human atria and ventricle. Ang 1-9 was liberated by a cathepsin A-type enzyme, Ang 1-7 by a different metallopeptidase-protease. Cathepsin A's presence in heart tissue was shown by deamidating enkephalinamide substrate, by immunoprecipitation and by immunohistochemistry. In immunohistochemistry, cathepsin A was detected in myocytes of atrial tissue. Ang 1-9 and Ang 1-7 potentiated the effect of an ACE-resistant bradykinin analogue on the B2 receptor in transfected cells expressing human ACE and B2, and in human endothelial cells. Ang 1-9 and 1-7 augmented arachidonic acid and NO release by bradykinin. NO liberation by bradykinin from endothelial cells was potentiated at 10nmol/L concentration by Ang 1-9 and Ang 1-7; at higher concentrations, Ang 1-9 was significantly more active. Both peptides had little activity in absence of bradykinin or ACE. Ang 1-9 and 1-7 potentiated bradykinin action on its B2 receptor at much lower concentrations than their IC50 values with ACE. They probably induce conformational changes in the ACE/B2 receptor complex via interaction with ACE.

Activation of bradykinin B1 receptor by ACE inhibitors.

ACE or kininase II inhibitors are very important, widely used therapeutic agents for the treatment of a variety of diseases. Although they inhibit ACE, thus, angiotensin II release and bradykinin (BK) inactivation, this inhibition alone does not suffice to explain their successful application in medical practice. Enalaprilat and other ACE inhibitors at nanomolar concentrations activate the BK B1 receptor directly in the absence of ACE and the peptide ligands, des-Arg-kinins. The inhibitors activate at the Zn-binding pentameric consensus sequence HEXXH (195 -199) of B1, a motif also present in the active centers of ACE but absent from the BK B2 receptor. ACE inhibitors, when activating the B1 receptor, elevate intracellular calcium [Ca2+]i and release NO from cultured cells. Activation by ACE inhibitor was abolished by Ca-EDTA, a B1 receptor antagonist, by a synthetic undecapeptide representing the 192-202 sequence in the B1 receptor, and by site-directed mutagenesis of H195 to A. With the exception of the B1 receptor blocker, these agents and the mutation did not affect the actions of the peptide ligand des-Arg10-Lys1-BK. Ischemia and inflammatory cytokines induce B1 receptors and elevate its expression. Direct activation of the B1 receptor by ACE inhibitors can contribute to their therapeutic efficacy, for example, by releasing NO in vascular beds, or to some of their side effects.

Kallikreins when activating bradykinin B2 receptor induce its redistribution on plasma membrane.

The bradykinin (BK) B2 receptor (R) is directly activated by kallikreins and other serine proteases independent of BK release. Both the Galpha(i) and Galpha(q) proteins are involved, shown by the release of arachidonic acid and [Ca2+]i elevation. Site-directed mutagenesis of the receptor and the lack of heterogeneous desensitization of the human B2R by the BK and kallikrein emphasize among others the differences between activation by the proteases and the peptide. To characterize further the mechanism thereby kallikreins activate and desensitize the B2R we investigated the distribution of the human B2R tagged with the green fluorescent protein (B2-GFP(Ct)) on the plasma membrane of stably transfected Chinese hamster ovary (CHO) cells. We visualized the movement of B2-GFP(Ct) R with confocal fluorescence microscopy after activation by BK or a by serine protease. Continued exposure of the cells to BK led to B2R internalization within 15-20 min. Porcine pancreatic and human recombinant tissue kallikreins induced a rapid definite redistribution of receptors on the plasma membrane within 5 min, prior to internalization. These effects of kallikrein were blocked by the B2R antagonist HOE 140 and by the kallikrein inhibitor, aprotinin. The B2R was also activated by endoproteinases LysC and ArgC and trypsin, but these enzymes did not induce redistribution, only internalization. In control experiments, kallikrein had no effect on cells transfected to stably express the angiotensin-converting enzyme-green fluorescent protein (GFP). Thus, kallikreins when activating the BK B2R also trigger its redistribution on plasma membrane.