Lateral Mobility of Integrin αIIbβ3 (Glycoprotein IIb/IIIa) in the Plasma Membrane of a Human Megakaryocyte

Abstract

SummaryThe migration of integrins to sites of cell-cell and cell-matrix contact is thought to be important for adhesion strengthening. We studied the lateral diffusion of integrin αIIbβ3 (glycoprotein Ilb/IIIa) in the plasma membrane of a cultured human megakaryocyte by fluorescence recovery after photobleaching of FITC-labelled monovalent Fab fragments directed against the P3 subunit. The diffusion of P3 on the unstimulated megakaryocyte showed a lateral diffusion coefficient (D) of 0.37 X10'9 cm2/s and a mobile fraction of about 50%. Stimulation with ADP (20 μM) or α-thrombin (10 U/ml) at 22° C induced transient decreases in both parameters reducing D to 0.21 X 10‘9 cm2/s and the mobile fraction to about 25%. The fall in D was observed within 1 min after stimulation but the fall in mobile fraction showed a lag phase of 5 min. The lag phase was absent in the presence of Calpain I inhibitor, whereas cytochalasin D completely abolished the decrease in mobile fraction. The data are compatible with the concept that cell activation induces anchorage of 50% of the mobile αIIbβ3 (25% of the whole population of receptor) to the cytoplasmic actin filaments, although, as discussed, other rationals are not ruled out.

Cytoplasmic regions of the β3 subunit of integrin αIIbβ3 involved in platelet adhesion on fibrinogen under flow conditions

“…Interference by cytochalasin D would release this constraint and shift the integrin to a conformation that is accessible to soluble fibrinogen molecules. Analysis of the mobility of α IIb β 3 by fluorescence recovery after photobleaching on a mature megakaryocyte also showed that cytochalasin D interferes with the immobilization of α IIb β 3 molecules induced by cell activation [29]. Our present data show that a similar mechanism might function in integrin α IIb β 3 binding surface‐bound fibrinogen.…”

The lateral mobility of NHE3 on the apical membrane of renal epithelial OK cells is limited by the PDZ domain proteins NHERF1/2, but is dependent on an intact actin cytoskeleton as determined by FRAP

“…3). These parameters for fluorescence recovery were similar to other plasma membrane proteins, including Na/K-ATPase (Paller, 1994), EGF receptor (Livneh et al, 1986), aquaporin-1 (Cho et al, 1999) and the integrin alpha IIb beta3 (Schootemeijer et al, 1997).…”

Agonist-specific Structural Rearrangements of Integrin αIIbβ3

“…1 Given its high segmental mobility, ␣ IIb ␤ 3 should adopt a large variety of shapes also in the membrane, where its lateral and rotational mobility has been measured (19,20). The integrin model that locates the subunit N-terminal domains at the globular head and the transmembrane and cytoplasmic domains at the tail ends was based on a combination of the morphological results and secondary structure prediction (21) and confirmed by the crystallographic structure.…”

“…The legs of ␣ IIb ␤ 3 are flexible and emerge from the same side of the head, with their tips either separated but never reaching a diametrical disposition (the head-free tail shapes), touching (the empty oval shapes) or overlapping (the bilobular shapes) each other, or fully folded on themselves and on the head (the filled globular shapes) (18). This high degree of flexibility of the tails of the heterodimer, seen in electron micrographs and deduced from the crystallographic structure, accounts for the short rotational correlation times measured in Triton X-100 solutions by time-resolved fluorescence anisotropy.1 Given its high segmental mobility, ␣ IIb ␤ 3 should adopt a large variety of shapes also in the membrane, where its lateral and rotational mobility has been measured (19,20). The integrin model that locates the subunit N-terminal domains at the globular head and the transmembrane and cytoplasmic domains at the tail ends was based on a combination of the morphological results and secondary structure prediction (21) and confirmed by the crystallographic structure.…”