Three discoveries were made during the current grant that serve as a basis for the present renewal: a) the identification of novel crosslinks which involve two post-translational modifications that stabilize collagen IV networks;b) identification of non-collagenous 1 (NC1) recognition domains governing the assembly of collagen IV networks;and c) identification of novel binding sites for integrins in collagen IV networks and a critical role for a1?1 and a2?1 integrins in modulating collagen IV homeostasis in kidney glomerulus in health and disease. Of particular importance, the recognition domains now provide us with a novel strategy to engineer, for the first time, the assembly of triple helical heterotrimers for studies of novel integrin binding sites in collagen IV networks. The central hypothesis of this renewal project is: On collagen IV networks, there are crosslink sites that stabilize the structure of these multivalent ligands, and distinct sites that confer binding of a1?1 and a2?1 integrins. In this renewal application, we will elucidate critical interactions at the molecular and atomic level of the two glomerular collagen IV networks and integrins. To test this hypothesis, we propose the following aims:
Aim 1) To deduce the chemical structure and the mechanism of formation of the novel hydroxylysine-methionine crosslink of collagen IV networks. We hypothesize that methionine and hydroxylysine residues are covalently connected through a sulfonium structure, and that the hydroxylation is catalyzed by one isoform of lysyl hydroxylase.
Aim 2) To map and characterize the novel a1?1 and a2?1 integrin binding sites in the a1a1a2 network. We hypothesize that a1a1a2 network of collagen IV possess distinct binding sites for alpl and a2p1 integrin.
Aim 3) To map and characterize integrin binding sites in the a3a4a5 network. We hypothesize that distinct binding sites for a1?1 and a2?1 integrin within a3a4a5 network could mediate specific glomerular functions. We anticipate that this study will generate novel insights into the structural basis whereby collagen IV networks bind integrins a1?1 and a2?1. This critical event is fundamental for the maintenance of glomerular function. These studies require a collaborative effort among integrin, collagen, cell, and structural biologists.
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