The interactions of cells of the vascular wall (endothelial cells, smooth muscle cells and fibroblasts) and platelets with collagenous components of the extracellular matrix exert profound influences upon cell phenotype affecting thrombosis, vessel wall repair, remodeling, inflammation, angiogenesis, and vasculogenesis. Indeed, fibrillar collagens have long been recognized as the most thrombogenic component of the blood vessel wall. Integrins represent the major class of cell surface receptors for extracellular matrix on these cells with the alpha2beta1 and alpha1beta1 integrins serving as receptors for native, triple helical collagens as established by studies in this and other laboratories. This proposal addresses three major unresolved issues in vascular cell collagen receptor biology.
The first aim employs a novel strategy using photoaffinity-labeled derivatives of the recombinant alpha1 and alpha2 integrin I domain (I domains represent independent, autonomously folding, highly homologous domains that are largely responsible for the ligand binding properties of the alpha2beta1 and alpha1beta1 integrins) to identify recognition sites for the alpha2beta1 and alpha1beta1 integrins on native, triple helical collagen molecules, an important issue heretofore unaddressable by approaches used to define simpler linear recognition sequences of other integrins. The structure(s) of the identified recognition sites will be confirmed and refined in additional studies employing synthetic, triple helical mini-collagens and recombinant mutant collagens and the intact alpha2beta1 and alpha1beta1 integrins. The second specific aim addresses the structural basis underlying the reciprocal type I vs. type IV collagen specificity/selectivity exhibited by the alpha2beta1 and alpha1beta1 integrins and their cognate I domains. This will be accomplished using recombinant chimeric I domains in which a single structural element (or part thereof) of one I domain has been replaced with that from the other and vice versa. Again key conclusions are validated with use of intact recombinant integrins containing the chimeric I domain. The third specific aim employs both biochemical and biophysical approaches to validate, extend and test predictions from the divalent cation displacement hypothesis of ligand binding to the alpha2 integrin I domain recently put forward by this laboratory. The multifaceted, innovative approaches outlined in this proposal offer the promise of significant advances in understanding the structure and function of collagen receptors and may serve as the structural foundation for ultimate pharmacologic intervention in collagen receptor pathobiology.
