Membrane type 1-matrix metalloproteinase: substrate diversity in pericellular proteolysis

Abstract

Enzymes in the matrix metalloproteinase (MMP) family have been linked to key events in developmental biology for almost 50 years. Biochemical, cellular and in vivo analyses have established that pericellular proteolysis contributes to numerous aspects of ontogeny including ovulation, fertilization, implantation, cellular migration, tissue remodeling and repair. Surface anchoring of proteinase activity provides spatial restrictions on substrate targeting. This review will utilize membrane type 1 MMP (MT1-MMP) as an example to highlight substrate diversity in pericellular proteolysis catalyzed by a membrane anchored MMP.

Fig. 1. Domain structure of MT1-MMP

(a) Mature membrane-anchored MT1-MMP is comprised of a catalytic domain (Y-G²⁸⁵) containing the Zn⁺⁺-binding consensus sequence, a hinge region (E²⁸⁶-I³¹⁸), hemopexin-like domain (C³¹⁹-C⁵⁰⁸), a membrane-adjacent stalk region (P⁵⁰⁹-S⁵³⁸), a transmembrane domain (A⁵³⁹-F⁵⁶²) and a cytoplasmic tail (R⁵⁶³-V⁵⁸²). (b) MT1-MMP undergoes autolytic processing at G²⁸⁴-G²⁸⁵ to generate a membrane-anchored species lacking the catalytic domain but retaining a surface-localized hemopexin domain that regulates activity of the mature enzyme. (c) MT1-MMP-catalyzed activation of proMMP-2 involves two molecules of MT1-MMP, likely functioning as a dimeric unit. One member of the complex forms a trimeric activation complex comprised of MT1-MMP, TIMP-2 and proMMP-2. The proMMP-2 in the trimeric complex is properly positioned for efficient activation by TIMP-2-free MT1-MMP, generating active MMP-2.

Fig. 2. Collagenolytic activity of MT1-MMP

(A, B) OvCa433 cells, with low endogenous MT1-MMP expression were transfected with either (A) a catalytically inactive (E240A mutant) or (B) wild type MT1-MMP and subcultured onto type I collagen gels containing quenched fluorescent type I collagen (DQ-collagen, Invitrogen). This collagen substrate is heavily conjugated with multiple fluorescein labels, leading to quenched fluorescence in the intact substrate. Upon hydrolysis to single dye labeled products, quenching is relieved yielding green fluorescent products indicative of collagenase activity. Note the pericellular collagenolysis in cells expressing wild type active MT1-MMP (B). (C) Invasion of type I collagen gels. OvCa433 cells transfected with wild type or catalytically inactive mutant (E240A) MT1-MMP or vector controls were seeded onto Transwell filters coated with a type I collagen gel and allowed to invade for 24 hr. Non-invading cells were removed from the upper chamber with a cotton swab prior to staining and enumeration of cells adherent to the underside of the filter using an ocular micrometer. Data from triplicate experiments are presented with S.D. value shown (*p<.005). (Figures courtesy of Yueying Liu.)

Fig. 3. Reciprocal regulation of MT1-MMP expression by collagen

Migrating cells encountering a three-dimensional collagen matrix engage collagen via integrin-mediated attachments. Clustering of integrins resulting from interaction with 3-dimensional collagen induces phosphorylation of Src kinases, resulting in induction of the transcription factor Egr-1 and transcriptional activation of the MT1-MMP promoter. The active proteinase is trafficked to the cell surface where it participates in pericellular collagenolysis.

Fig. 4. MT1-MMP processing of cell surface substrates

MT1-MMP catalyzes limited proteolytic processing and/or ectodomain shedding of a variety of membrane-anchored molecules including cadherins, integrins, proteoglycans (PG) and PG receptors as well as other surface substrates including LRP, RANKL, semaphoring 4D etc. These cleavages have been shown to regulate diverse cellular processes as summarized above.