Highly stoichiometric, stable, and specific association of integrin alpha3beta1 with CD151 provides a major link to phosphatidylinositol 4-kinase, and may regulate cell migration

Abstract

Here we describe an association between alpha3beta1 integrin and transmembrane-4 superfamily (TM4SF) protein CD151. This association is maintained in relatively stringent detergents and thus is remarkably stable in comparison with previously reported integrin-TM4SF protein associations. Also, the association is highly specific (i.e., observed in vitro in absence of any other cell surface proteins), and highly stoichiometric (nearly 90% of alpha3beta1 associated with CD151). In addition, alpha3beta1 and CD151 appeared in parallel on many cell lines and showed nearly identical skin staining patterns. Compared with other integrins, alpha3beta1 exhibited a considerably higher level of associated phosphatidylinositol-4-kinase (PtdIns 4-kinase) activity, most of which was removed upon immunodepletion of CD151. Specificity for CD151 and PtdIns 4-kinase association resided in the extracellular domain of alpha3beta1, thus establishing a novel paradigm for the specific recruitment of an intracellular signaling molecule. Finally, antibodies to either CD151 or alpha3beta1 caused a approximately 88-92% reduction in neutrophil motility in response to f-Met-Leu-Phe on fibronectin, suggesting an functionally important role of these complexes in cell migration.

Figure 1

Among β1 integrins, α3β1 has the most associated PtdIns 4-kinase activity. (A) K562 transfectants were lysed in 1% Brij 99 and immunoprecipitated with anti-β1 (left panel) or anti-CD81 (right panel) antibodies (TS2/16 and M38, respecely). Immunoprecipitates were then assayed for specific lipid kinase activity as described in MATERIALS AND METHODS. Data are representative of three separate experiments. (B) Lipid kinase activity was measured in immunoprecipitates from HT1080 cells. HT1080 cells express approximately 138, 167, 91, 100, 55, and 245 MFI units of α2, α3, α5, α6, αv, and CD98, respectively. Data are representative of four separate experiments. (C) K562 cells transfected with wild-type α3 or chimeric α3 (X3TC5) were lysed in 1% Brij 99 and immunoprecipitated with anti-β1, before determining lipid kinase activity. Data are presented as the mean ± SD from three separate immunoprecipitations and are representative of two experiments.

Figure 2

CD151 specifically associates with α3β1. (A) HT1080 fibrosarcoma cells were lysed with either 1% Brij 96 (upper panel) or 1% Triton X-100 (lower panel) before immunoprecipitation with the indicated antibodies. The immune complexes were resolved by nonreducing SDS-PAGE, immunoblotted with anti-β1 mAb TS2/16, and developed by chemiluminescence. HT1080 cells express approximately 325, 33, 94, and 68 MFI units of β1, MHC class I, CD151, and CD81, respectively. (B) Metabolically labeled HT1080 cells were lysed in 1% Brij 96 supplemented with 0.2% SDS and immunoprecipitated with the indicated antibodies. Immune complexes were eluted from protein A-Sepharose at low pH (pH 2.7) and either directly analyzed (lanes a–d) or reimmunoprecipitated with 5C11-Sepharose (lanes e–g) in 1% Brij 96 containing 0.5% SDS, as described in MATERIALS AND METHODS. Fourfold more immunoprecipitated material was loaded in lane d compared with lanes a–c. Immunoprecipitates were resolved by 12% nonreducing SDS-PAGE, and proteins were detected by autoradiography. (C) K562 cells transfected with the indicated α chain cDNAs or vector-alone (lane e) were lysed in 1% Triton X-100, and lysates were immunoprecipitated with antibodies against CD151 (5C11, upper panel) or β1 (TS2/16, lower panel). Immune complexes were resolved by SDS-PAGE and immunoblotted with anti-β1 mAb TS2/16. K562-X3TC5 cells (lane c) are transfected with a chimeric α3 containing the α3 extracellular domain and α5 transmembrane and cytoplasmic domains.

Figure 3

CD151–α3β1 complexes are biochemically stable and form before cell lysis. (A) Metabolically labeled HT1080 cells were lysed and immunoprecipitated with 5C11 mAb in 2% n-octyl-β-d-glucopyranoside alone (lane e) or supplemented with 10 mM EGTA (lane a), 10 mM EDTA (lane b), 1 M LiCl (lane c), or 100 mM Tris pH9.0 (lane d). (B) The cell lines K562, K562-α3, and CHO-α3β1 (containing human α3 and β1) were lysed in 1% Triton X-100, mixed 1:1 with K562 cell lysate or lysis buffer alone, and subsequently immunoprecipitated with antibodies against β1 (TS2/16), CD63 (6H1), or CD151 (5C11). Immune complexes were resolved by 8% nonreducing SDS-PAGE and immunoblotted with an antibody against β1 (TS2/16).

Figure 4

Transfection of α3 increases the cell surface expression of CD151. K562 cells transfected with α2, α3, α4, or α6 cDNA, or vector alone (K562-α5(Neo)) were stained with the negative control antibody, P3 (dotted lines), or with antibodies against β1 (TS2/16, first column), CD63 (6H1, second column) or CD151 (5C11, third column) before analysis by flow cytometry. Specific mean fluorescence intensity (MFI) values are indicated. Except for K562-α5(Neo) (in which α5β1 is the only β1 integrin), transfectants express similar levels of the respective α chains. The transfected α chains represent between 72 and 94% of total β1 integrin in these transfectants.

Figure 5

CD151 and α3β1 codistribute in the basal layer of keratinocytes of human epidermis. Sections of human skin were stained with mAbs against α3 (A and C), CD151 (B), or CD9(D), as described in MATERIALS AND METHODS. Arrowheads point to the basal keratinocytes. E, epidermis; D, dermis. No staining was observed using secondary antibodies alone (our unpublished results).

Figure 6

Analysis of α3β1-CD151 stoichiometry. Metabolically labeled HT1080 cells were lysed in 1% Triton X-100, and lysates were cleared using anti-CD151 5C11-conjugated Sepharose (+) or unconjugated Sepharose (−). Lysates were subsequently immunoprecipitated with the indicated antibodies, and immune complexes were resolved by nonreducing SDS-PAGE before detecting integrin α chains (A), CD81 (B), or CD151 (C) by autoradiography. The percent reduction in the levels of integrin immunoprecipitated upon CD151 immunodepletion was determined by measuring the amount of ³⁵S-methionine corresponding to the respective proteins in a Betascope 603 Blot Analyzer. At least 400 counts of ³⁵S-methionine were detected in all positive (i.e., nondepleted) lanes during 1 h; and background (< 10%) was subtracted before dividing CD151 depleted/nondepleted counts.

Figure 7

PtdIns 4-kinase is present in CD151–α3β1 complexes. (A) HT1080 cells were lysed in 1% Brij 99 and immunoprecipitated with the indicated antibodies. Immunoprecipitates were assayed for kinase activity as described in MATERIALS AND METHODS. The monoclonal antibodies A2-IIE10 (α2), A3-IVA5 (α3), 5C11 (CD151), and 6H1 (CD63) were used for immunoprecipitation. (B and C) Cell lysates were immunodepleted using unconjugated Sepharose beads, or beads conjugated with anti-CD151 mAb 5C11, anti-CD63 mAb 6H1, anti-CD81 mAb M38, or combined anti-CD63/anti-CD81 mAbs, before immunoprecipitation of α3 (B) or CD63, CD81, and CD151 (C). Immunoprecipitates were assayed for lipid kinase activity, and results are expressed as percent reduction of specific lipid kinase activity resulting from immunodepletion with CD151, CD63, CD81, or CD63/CD81 combined. Results each represent the mean of 4–5 experiments (1 using A431 cells, 3–4 using HT1080 cells).

Figure 8

Inhibition of PMN chemotaxis by antibody perturbation of α3β1 and CD151. PMN migration in response to fMLP was measured on fibronectin in the presence of β-glucan. mAbs A3-X8 (α3), 5C11 (CD151), C9-BB(CD9), 6H1 (CD63), 8A6 (CD98), and 4.6.19 (CD32) were incorporated into the agarose, as described in MATERIALS AND METHODS. Isotype-matched IgG and IgM served as controls. None of these antibodies triggered neutrophil homotypic aggregation. Migration distances, calculated from Microprojector-magnified images, are presented as mean ± SD from quadruplicate samples. Data are representative of three separate experiments. Background random migration values (no fMLP) ranged between 3–6 mm. mAbs against α3 and CD151 significantly inhibited PMN motility (p < 0.0001).