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

Abstract

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.

Figure 1. Resensitization of B₂-GFP receptors to bradykinin by enalaprilat (EPT)

After CHO/ACE+B2-GFP cells were stimulated and desensitized with 1μM bradykinin (BK) (A) or 1μM bradykinin analogue (BKan) (B) for 30 min at 37°C, then they were treated with either medium alone, a second dose of 1μM BK (A) or 1M BKan (B), or 1μM EPT for 5 min at room temperature. Ordinate: relative amount of [³H] arachidonic acid ([³H]AA) released; Baseline = 1. Data are mean ± SEM. In (A), n=4, *, p<0.01; compared with 1μM BK treatment; in (B); n=4, **, p<0.005, compared with 1μM BKan treatment. R.U. = relative unit. The second dose of agonist added to desensitized cells in absence of EPT was inactive. EPT sensitized the B₂ receptors to agonist in the medium.

Figure 2. Coimmunoprecipitation of B₂-GFP and ACE in CHO/ACE+B₂-GFP cells

A: Immunoblotting with anti-GFP antiserum (1:2,000 v/v). Attempts for immunoprecipitates (IP) of S2 lysates from various types of cells: CHO (WT-CHO), CHO/ACE, CHO/B2-GFP (CHO/BG) and CHO/ACE+B₂-GFP (CHO/ABG) cells. Antiserum to ACE precipitated the protein only from the S2 cell lysates of cells that express ACE, and anti-GFP reacted with the coprecipitate of ACE and B₂-GFP in a Western blot of CHO-ABG cells. B: Proteins immunoblotted on gels with anti-ACE antiserum (1:4,000 v/v) after immunoprecipitation with anti-GFP. The antisera were applied in reverse order, but conditions were the same as in A. Note that the molecular mass of human ACE is about 180 kDa and of B₂-GFP is about 100 kDa. ACE and B₂ were coprecipitated with antisera either to human ACE or GFP when they were from transfected cells expressing both human ACE and B₂ bradykinin receptors.

Figure 3. Colocalization of ACE and B₂-GFP in fixed CHO/ACE+B₂-GFP cells

The green color is due to GFP, and the red to Alexa 568 conjugated secondary IgG that recognizes the primary antibody against ACE. A: B₂-GFP (green, left) and ACE (red, middle), are colocalized (yellow, right). B, upper: Enlarged view of panel A. B, lower: cross-sectional images of cells viewed in the vertical (Z) plane showing extensive colocalization (yellow) on the cell membrane.

Figure 4. FRET between ACE and B₂ receptors

Confocal microscopy of CHO/ACE-CFP+B₂-YFP cells shows a 2 μm optical slice in an x,y plane through the middle of a CHO cell coexpressing ACE-CFP and B₂-YFP. Linear unmixing of CFP and YFP emission spectra was done with the Zeiss LSM-510 META detector. Pre-bleach images were collected (A, B, C; note region of interest [ROI] outlined in white; Bar = 10 μm). YFP fluorescence was photo-bleached in the ROI by scanning with a 514 nm laser, and post-bleach images were collected (D, E, F). Note increase in donor intensity (A and D) following bleaching of receptor (B and G). This representative experiment of 9 indicates energy transfer (FRET) between the donor ACE enzyme and the acceptor, B₂ receptor, and it follows that ACE and B₂ receptors colocalize within 10 nm.

Fig 5. Bradykinin stimulates movements of ACE and B₂ receptors

CHO/ACE-GFP+B₂-mRFP cells. A. ACE-GFP and B₂-mRFP are diffusely distributed on the cell membrane prior to adding an agonist. B. Within one min after 1 μM BKan was added, ACE and B₂ receptors have begun to colocalize on the cell surface membrane (arrowheads). C. At two min after stimulation, numerous patches of colocalized ACE and B₂ receptor are evident on the surface (arrowheads). D. Within ten min, ACE and B₂ receptors are largely divergent from the colocalized patches (*arrowheads, compared to areas in C). E. Thirty min after stimulation, colocalized ACE and B₂ receptors appear once again diffusely distributed at the surface. At this time, co-internalization of ACE and B₂ is also evident (see Fig 6). Bar = 20 μm.

Fig 6. Internalized ACE-GFP and B₂-mRFP

CHO cells were fixed 30 min after stimulation with 1 μM BKan. ACE and B₂ appear colocalized (yellow) and cointernalized from the plasma membrane. Bar = 10 μm.

Fig 7. Lack of potentiation and resensitization in CHO cells expressing ACE-B₂ receptor fusion protein

A: Receptor (R) function. Cells were stimulated with 10 nM bradykinin in absence or in presence of HOE 140 and the released [³H] AA was measured after 30 min. Bradykinin stimulated a 3 fold increase in AA release that was blocked by HOE 140, indicating that the receptors in the fusion protein were functional. (*p < 0.001 v. medium control, n = 6). B: Lack of potentiation. Cells were exposed to 50 or 100 nM BKan with or without pretreating them with enalaprilat (EPT). The same relative amount of AA was released is in the presence or absence of EPT. HOE 140 (1 μM) blocked the effect of BKan on B₂ receptors (*, p < 0.001 v. medium control, n = 6). C: Lack of resensitization. Cells were pre-incubated with 1 μM BKan for 30 min at 37°C to desensitize the receptors. Then medium or 1 μM BKan or 1 μM EPT was added for 5 min at 37°C. The second dose of BKan did not stimulate the release of AA, indicating that the receptors were desensitized. EPT did not stimulate AA release, thus it did not resensitize the ACE- B₂ fusion receptors to BKan in the medium (NS, not significant v. medium control, n = 9, R.U. = relative unit). In the ACE-B₂ fusion protein, where only the receptors are attached to the plasma membrane, but not ACE; ACE inhibitor neither potentiated bradykinin nor resensitized the B₂ receptors fused to it, although both the B₂ receptor and ACE (see Results) remained active.