Activation of alpha(v)beta3-vitronectin binding is a multistage process in which increases in bond strength are dependent on Y747 and Y759 in the cytoplasmic domain of beta3

Abstract

Integrin receptors serve as mechanical links between the cell and its structural environment. Using alpha(v)beta3 integrin expressed in K562 cells as a model system, the process by which the mechanical connection between alpha(v)beta3 and vitronectin develops was analyzed by measuring the resistance of these bonds to mechanical separation. Three distinct stages of activation, as defined by increases in the alpha(v)beta3-vitronectin binding strength, were defined by mutational, biochemical, and biomechanical analyses. Activation to the low binding strength stage 1 occurs through interaction with the vitronectin ligand and leads to the phosphorylation of Y747 in the beta3 subunit. Stage 2 is characterized by a 4-fold increase in binding strength and is dependent on stage1 and the phosphorylation of Y747. Stage 3 is characterized by a further 2.5-fold increase in binding strength and is dependent on stage 2 events and the availability of Y759 for interaction with cellular proteins. The Y747F mutant blocked the transition from stage 1 to stage 2, and the Y759F blocked the transition from stage 2 to stage 3. The data suggest a model for tension-induced activation of alpha(v)beta3 integrin.

Figure 1

Spinning disk comparison of Kα_vβ3 (A), Kα_vβ3(Y747F) (B), and Kα_vβ3(Y759F) (C) cells, with and without PMA activation. Data from two different discs are plotted on the same axis. ●, control data points; ○, control curve fit; ▪, PMA data points; □, PMA curve fit. Lines represent sigmoid curve fits.

Figure 2

Stage 1 activation of α_vβ3. (A) Adhesion of Kα_vβ3(Y747F) cells to vitronectin were plated for 15 min on either BSA (●) or vitronectin-coated coverslips (coated at 2 μg/ml; ▪) and analyzed using the spinning disk. Solid lines represent the curve fits for each data set. The adhesion strength is defined as the force required to detach 50% of the cells. Adhesion strength for BSA = 6.4 ± 1.65 dynes/cm² (means ± SD; n = 6). Adhesion strength for vitronectin = 23.9 ± 3.3 dynes/cm² (n = 8). (B) Specificity α_vβ3(Y747F)-mediated adhesion. Data show adhesion strength and SD derived from cell detachment profiles as shown in A: BSA cells plated on BSA only, Vn1 cells plated on vitronectin (1 μg/ml), AIIB2 cells preincubated with antibody to β1, and LM609 cells preincubated with antibody to β3.

Figure 3

Cross-linking of activated α_vβ3: αv and β3 subunits. Kα_vβ3 and Kα_vβ3(Y747F) cells were plated on vitronectin (2 μg/ml) coated plates for 15 min and cross-linked with sulfo-BSOCOES for 5 min. Extract lanes show that α_v and β3 levels were unaffected by either PMA or cross linking. The X-linked lanes represent the levels of cross-linked α_v and β3 recovered after cleavage of the cross-linker. Treatment of Kα_vβ3 cells with PMA increased the level of cross-linked α_v and β3, demonstrating a change in the α_vβ3-vitronectin binding conformation.

Figure 4

Adsorption of vitronectin to glass. ¹²⁵I labeled-vitronectin was adsorbed to glass using the same procedures used to coat glass coverslips for the spinning disk assays. Above 3 μg/ml coating concentration, the vitronectin saturated the surface, reaching a plateau value of 100 ng/cm² (plateau data points are not shown). Error bars, SD; solid line is a linear regression.

Figure 5

Comparison of surface expression of β1 and β3 integrin on transfected K562 cells. For FACS analysis the control is secondary antibody alone; LM609 was used to quantify β3 integrin, and AIIB2 was used to quantify β1 integrin. Mean fluorescence index (MFI) was calculated: [(geometric mean of the positive fluorescence) − (geometric mean of the control)]/(geometric mean of the control).

Figure 6

Analysis of adhesion strength constants for different binding states of α_vβ3. Kα_vβ3 (A), Kα_vβ3(Y747F) (B), and Kα_vβ3(Y759F) (C) cells were analyzed using the spinning disk device on different vitronectin densities for 15 min of plating: control (●) or PMA-treated (○). The adhesion strength was determined from the sigmoid curve fits for each determination and plotted as a function of vitronectin density. Dashed line, the background adhesion, is not subtracted from the other plotted values. Figure shows one representative of four experiments.

Figure 7

The relative binding strength constants To calculate the relative binding strength, the expression levels of α_vβ3 were normalized to the level of expression on the K α_vβ3 cells based on the mean fluorescence index in Figure 5. The initial slope of the plots in Figure 6 were divided by the relative expression level of β3 integrin to give the relative binding strength constants. Background nonspecific adhesion has not been subtracted.

Figure 8

Adhesion Strength for α_vβ3, α_vβ3(Y747F), and α_vβ3(Y759F) expressed in CS1 melanoma cells. The adhesion strength for each cell line was measured for vitronectin coating of 30 ng/cm² in a 10-min incubation. The measured adhesion strengths were normalized for the level of β3 expression using the mean fluorescent index as measured by FACS.

Figure 9

Adhesion strength after α_vβ3 cross-linking to vitronectin. The adhesion strength (▪) and the cross-linked adhesion strength (□) after chemical cross-linking of α_vβ3 to substrate-bound vitronectin were determined using the spinning disk analysis to Kα_vβ3, Kα_vβ3(Y747F), and Kα_vβ3(Y759F) cells seeded on vitronectin (60 ng/cm²) for 15 min in the absence or presence of PMA. The α_vβ3-cytoskeletal apparent binding strengths were approximately twice the corresponding α_vβ3-vitronectin binding strengths.

Figure 10

Phosphorylation of β3 integrin. Kα_vβ3, Kα_vβ3(Y747F), and Kα_vβ3(Y759F) cells were held in suspension in the presence or absence of PMA or plated for 2 h on vitronectin-coated plates. Blots were first probed for phosphotyrosine (top), stripped, and then reprobed for β3 integrin (bottom). Binding of cells to vitronectin-stimulated phosphorylation of β3 but PMA did not. No phosphorylation of β3 was seen for the Y747F mutant and 14% of WT levels of phosphorylation were observed for the Y759 mutant.

Figure 11

Model for activation of α_vβ3. Four states for α_vβ3 integrin are given each with a different interaction with the vitronectin ligand from unbound to high-strength binding and with different linkages to the actin cytoskeleton. Y, available tyrosines; P, phosphorylated tyrosines. Diamonds and pentagons represent actin–integrin linking proteins, which may include talin and a-actinin. Actin arrows indicate the application tension by the actin cytoskeleton.