VPS33B, a Member of the Sec1/Munc18 Protein Family, Binds to Integrin Beta Subunits and Is Required for α_IIbβ₃ Outside-in Signaling

Integrins play fundamental roles in many biological processes such as development, immunity, cancer, wound healing, hemostasis, and thrombosis. Integrin activation is essential for cell adhesion, spreading, survival, proliferation, and migration. Integrins are heterodimeric transmembrane glycoproteins composed of a and b subunits. The function of integrins is modulated by bi-directional transmembrane signaling: inside-out and outside-in signaling, which are mediated through the interactions between integrin a or b subunit cytoplasmic tails and intracellular proteins and can be regulated by many different biochemical signaling pathways. α_IIbβ₃ is a major integrin expressed in megakaryocytes and platelets. Antagonists of α_IIbβ₃ are potent anti-thrombotic drugs and new inhibitors targeting α_IIbβ₃ are under preclinical testing or large patient trials to treat acute coronary syndromes. Integrin α_IIbβ₃ activation involves binding of proteins, including talin, kindlins, Src kinase, and Gα₁₃ to the cytoplasmic domain of its β subunit. To gain insight into α_IIbβ₃signaling and to identify new proteins that regulate α_IIbβ₃ activation, agarose beads conjugated with glutathione S-transferase (GST)-β₃ integrin cytoplasmic domain fusion protein (GST-β₃CD) or GST-α_IIb integrin cytoplasmic domain fusion protein (GST-α_IIbCD) were incubated with human platelet lysates. Proteins pulled down by the GST-β₃CD or GST-α_IIbCD beads were subjected to mass spectrometric analysis. We found that the GST-β₃CD but not the GST-α_IIbCD beads specifically pulled down a previously unreported protein, the vacuolar protein sorting-associated protein 33B (VPS33B), encoded by the VPS33B gene. To verify that VPS33B is in a complex with α_IIbβ₃, we infected Chinese hamster ovary (CHO) cells stably expressing α_IIbβ₃ (A5 cells) with an adenovirus containing FLAG epitope-tagged human VPS33B cDNA. Cells were allowed to adhere and spread on fibrinogen. FLAG-VPS33B exhibited a significant intracellular colocalization with a_IIbb₃. Immunoblotting analysis revealed a specific association of b₃ with precipitated FLAG-VPS33B. The specific interaction was also confirmed by reciprocal immunoprecipitation using a specific β₃antibody. Furthermore, in an in vitro binding assay, we were able to pull down VPS33B from the lysates of CHO cells overexpressing VPS33B by the GST-β₃CD beads. The interaction appears to be direct, since we were also able to pull down purified FLAG-VPS33B with GST-β₃CD and GST-β₁CD proteins. The pulldown assay showed that the β₃ fragment spanning residues 716~730 bound efficiently to FLAG-VPS33B. In contrast, the fragments containing either residues 729~762 or residues 744~762 fragments failed to do the same. More importantly, using the rabbit polyclonal antibody against VPS33B, we were able to coimmunoprecipitate endogenous b₃ subunit with VPS33B from human platelet lysate. Thus, we identified a novel binding protein of integrin β₃.

Next, we investigated the role of VPS33B in integrin activation using a recombinant integrin activation model of CHO cells. Overexpression of VPS33B in CHO cells expressing α_IIbβ₃ (A5 cells) markedly potentiated cell spreading on fibrinogen and F-actin formation. To establish a role of VPS33B in integrin activation in platelets, we created a mouse model with megakaryocyte- and platelet-specific deletion of VPS33B. Platelets lacking VPS33B were defective in spreading on fibrinogen. VPS33B^-/- platelets failed to support clot retraction. On the other hand, thrombin-induced fibrinogen binding to platelets and platelet aggregation were not affected by the loss of VPS33B. Collectively, these results demonstrate an essential role of VPS33B in α_IIbβ₃ outside-in signaling but is not requried for integrin inside-out signaling. We further demonstrate that VPS33B promotes α_IIbβ₃ outside-in signaling through RhoA and Rac1 activation, leading to clot retraction and cell spreading, respectively. Therefore, our results for the first time establish vesicle trafficking proteins as an important novel class of modifiers of integrin function in platelets and cells.