Integrins are ?/? heterodimeric cell surface receptors, which, via their ability to exist in multiple conformations, regulate diverse biological processes and play critical roles in many human diseases. Proteins binding to either the extracellular or cytoplasmic face of an integrin can effect long-range conformational changes that convert integrin from a resting low-affinity to an activated high-affinity state, a tightly regulated process for integrins such as ?IIb?3 that is essential for both hemostasis and thrombosis. Normal biological functions of integrins are regulated by a tightly-controlled balance between activated and deactivated states. Although much progress has been made in understanding the mechanisms and molecules involved in integrin activation, the process of integrin deactivation is poorly understood. We have been engaged in structural and functional analyses of ligand-binding-induced integrin conformational changes leading to activation and have identified a novel series of competitive inhibitors, which, unlike conventional inhibitors, ca displace ligand from ?IIb?3 without inducing conformational changes. Based on these findings, this grant seeks to examine the structural transitions that occur during integrin deactivation, which we hypothesize is governed by intrinsic structure features of the integrin. A combination of novel crystallographic, biochemical and biophysical approaches will be used to define distinct stages of conformational deactivation of the ?IIb?3 headpiece at the atomic level, in intact integrins on the cell surface, and in solution. The relative contributions of specific interactions among the extracellular, transmembrane, and cytoplasmic domains in integrin deactivation will be defined. The importance of N-linked glycosylation in the conformational regulation of ?IIb?3 and ?V?3 integrins will be characterized and correlated with aberrant integrin glycosylation in human diseases like cancer and diabetes. Outcomes of these studies will enable us to explore novel and widely applicable strategies for designing integrin antagonists that block ligand binding while stabilizing (rather than disrupting) the inactive conformation and which may, therefore, be superior to existing integrin antagonists, which inadvertently induce integrin activation.
A second aim focused on the role of ? integrin cytoplasmic tail (CT) in integrin function, grew out of our recent observation that the membrane-distal (MD) region of the ?-integrin CT is indispensable for integrin activation. Using ?IIb, ?L, and ?5 integrins as platfoms for generating MD region swapped chimeras, we will examine the sequence and structural determinants of the ? integrin CT MD region in regulating integrin conformational activation and signaling. Using multifaceted approaches, we will examine the conformational changes of both ? and ? transmembrane and cytoplasmic domains during integrin activation in situ. Together, these complementary Specific Aims will advance our understanding of the molecular basis for integrin conformational transitions and provide structure-based insights into bidirectional integri signaling, which will facilitate the development of novel integrin antagonists that have improved therapeutic efficacy.
Integrins are a large family of cell surface molecules that are involved in the progression of many diseases, including thrombosis, inflammation, and cancer, and thus are important therapeutic targets. The platelet integrin ?IIb?3 is essential in platelet platelet interactions required for preventing bleeding and abnormal thrombotic disorders such as heart attacks and stroke. Using ?IIb?3 integrin as a model molecule, this project proposes to address issues critical to understanding the activation, structural regulation, and pharmacologic inhibition of clinically-relevant members of the integrin family.
