Proteolysis of the extracellular matrix is highly regulated. An illustrative example of physiologically relevant spatial regulation of proteolysis is seen with matrix metalloproteinase 1 (MMP-1, collagenase 1) in human cutaneous wounds. As initially established by members of this group, in response to injury, MMP-1 expression is uniquely induced in basal epidermal cells (keratinocytes) by contact with type I collagen in the dermis as they move off the basement membrane. The inductive stimulus provided by collagen is mediated by the collagen binding integrin, alpha2beta1. Furthermore, the catalytic activity of MMP-1 is required for keratinocyte migration on collagenous matrices. Thus, MMP-1, collagen and the alpha2beta1integrin function together in migrating keratinocytes. We have recently established that MMP-1, but not other MMPs, specifically and selectively binds to the alpha2beta1integrin. The physical and functional associations of MMP-1 with the (2(1 suggest the overall guiding hypothesis that binding of MMP-1 to the alpha2beta1integrin is an important mechanism by which cell surface MMP-1 activity is regulated.
The aims of this proposal are directed at elucidating the structural basis and functional consequences of MMP-1 binding to the alpha2beta1 integrin.
Specific aim 1 will employ an iterative mutagenesis strategy to define the structural determinants of the alpha2beta1 integrin subunit I domain required for the binding of MMP-1. Preliminary data support the hypothesis that binding of MMP-1 to the integrin is mechanistically distinct from that of other alpha2beta1 ligands. In complementary studies, specific aim 2 will employ an iterative chimeragenesis strategy to define the structural determinants within the hemopexin-like and linker domains of MMP-1, which, as established in preliminary studies, are required for binding to the alpha2beta1 integrin.
Specific aim 3 will employ biochemical and cell biologic approaches to test three on-mutually exclusive hypotheses regarding the mechanism(s) by which binding of MMP-1 to the alpha2beta1 integrin regulates cell surface MMP-1 activity: 1) proMMP-1 binding to the alpha2beta1 integrin facilitates conformational (non-proteolytic) activation of the MMP, 2) proMMP-1 binding to the (2(1 integrin facilitates proteolytic activation of the MMP by other proteinases, and 3) binding of MMP-1 to the (2(1 integrin diminishes the susceptibility of MMP-1 to inhibition by TIMP-I.
