Regulation of class A scavenger receptor-mediated cell adhesion and surface localization by PI3K: identification of a regulatory cytoplasmic motif

Abstract

The importance of cytoplasmic motifs in differentially regulating SR-A function was demonstrated by deleting the first 49 cytoplasmic aa (SR-A(Delta1-49)), which abolished SR-A-mediated ligand internalization without reducing cell adhesion. To identify additional cytoplasmic motifs within the first 49 aa that regulate SR-A function, the acidic residues in a conserved motif (EDAD) were changed to their amide derivatives (SR-A(QNAN)). The function and regulation of SR-A(QNAN) were compared with that of SR-A(Delta1-49) and SR-A in transfected HEK-293 cells. Blocking PI3K activation inhibited SR-A, but not SR-A(Delta1-49)- or SR-A(QNAN)-mediated cell adhesion. Although deleting (SR-A(Delta1-49)) or mutating (SR-A(QNAN)) the EDAD motif abolished the PI3K sensitivity of SR-A-mediated cell adhesion, these mutations did not affect ligand internalization or PI3K activation during cell adhesion. To define the mechanism by which PI3K regulates SR-A-mediated cell adhesion, the cellular localization of wild-type and mutant SR-A was examined. PI3K inhibition reduced surface localization of SR-A but not of SR-A(Delta1-49) or SR-A(QNAN). The regulation of SR-A surface localization by PI3K was confirmed in peritoneal macrophages, which endogenously express SR-A. Together, these results suggest a pathway in which SR-A binding to an immobilized ligand activates PI3K to recruit more receptor to the plasma membrane and enhances cell adhesion.

Figure 1.

An acidic aa motif is required for PI3K-dependent, SR-A-mediated cell adhesion but not ligand uptake. (A) The aa sequence of the SR-A cytoplasmic tail for SR-A constructs (SR-A_, SR-A_QNAN, and SR-A_Δ1–49) used in this study. (B and C) Cell attachment and spreading assays of HEK cells expressing different SR-A constructs and pretreatment with wortmannin (PI3K inhibitor) were performed as described in Materials and Methods. Cell morphology (B) was assessed by staining with Alexa-Fluor⁵⁶⁸-conjugated phalloidin (red) and DAPI (blue). Images (40× original) are representative of at least five independent experiments. The original scale bars represent 50 μm. Cell attachment (C) was determined using the CyQuant assay. The number of cells adhered was quantified and expressed as a percentage of total cells plated. Values are expressed as mean ± sem; n = 4; *, P < 0.05, compared with control SR-A values. (D) SR-A-mediated ligand uptake was assessed as described in Materials and Methods. The means ± sem of three individual experiments are shown. *, P < 0.05, compared with SR-A control values.

Figure 2.

Expression and PI3K activation during adhesion of transfected HEK-293 cells. Transfected HEK cells were cultured in suspension with tetracycline (0.5 μg/ml, 16 h) to induce SR-A expression. (A) Induced cells were adhered for 120 min, cell lysates were prepared, and proteins were resolved by SDS-PAGE under reducing and nonreducing conditions and then immunoblotted with a SR-A antibody. The arrows indicate the monomeric (≈70 kDa), dimeric, and trimeric forms of the receptor. (B) Induced cells were kept in suspension (0 min; susp) or allowed to adhere for 30 or 120 min. Cell lysates were prepared, and proteins were resolved by SDS-PAGE and immunoblotted for phospho-S⁴⁷³Akt (pAkt) and total Akt (tAkt). Blots are representative of at least three independent experiments.

Figure 3.

An acidic aa motif is required for PI3K-dependent SR-A cell surface localization. Following SR-A induction and treatment with PI3K inhibitors in suspension, cells were allowed to adhere for 2 h. Cell surface proteins were biotinylated and precipitated from cell lysates using streptavidin-coated beads as described in Materials and Methods. The amount of precipitated (Surface) SR-A and total SR-A in cell lysates was assessed via immunoblotting with antibodies specific for SR-A. The intensities of surface SR-A were normalized to that detected in control SR-A-expressing cells. The amount of GAPDH in cell lysates was blotted as a control. Results obtained from a representative experiment and the mean ± sem of three separate experiments are shown. *, P < 0.05, compared with control SR-A values.

Figure 4.

PI3K activation increases SR-A surface localization during macrophage adhesion. MPM were isolated, treated in suspension with wortmannin, and then kept in suspension or allowed to adhere for 2 h. (A) Cell surface proteins were biotinylated and precipitated from lysates as described for Figure 3. The amount SR-A present in the original lysate, streptavidin precipitate (surface), or supernatant (intracellular) was assessed via immunoblotting with antibodies specific for SR-A and the intensities normalized to that detected in control macrophages kept in suspension. Results obtained from a representative experiment and the mean ± sem of four separate experiments are shown. *, P < 0.05, compared with control suspension values. (B) Alternatively, the amount of SR-A on the cell surface was determined labeling surface receptors with Alexa⁴⁸⁸-conjugated 2F8 mAb (30 min, 4°C) and cell-associated fluorescence quantified by flow cytometry. Data from a representative experiment are shown. The blue tracing indicates 2F8 association with macrophages in suspension, the black tracing indicates 2F8 association following 120 min of adhesion, and the red tracing indicates 2F8 association with LY294002-treated macrophages that were adhered for 120 min.