Search

Matrix metalloprotease (MMP) activation contributes to the degradation of the extracellular matrix (ECM), resulting in a multitude of pathologies. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifaceted endocytic and signaling receptor that is responsible for internalization and lysosomal degradation of diverse proteases, protease inhibitors, and lipoproteins along with numerous other proteins. In this study, we identified MMP-1 as a novel LRP1 ligand. Binding studies employing surface plasmon resonance revealed that both proMMP-1 and active MMP-1 bind to purified LRP1 with equilibrium dissociation constants (KD) of 19 and 25 nM, respectively. We observed that human aortic smooth muscle cells readily internalize and degrade 125I-labeled proMMP-1 in an LRP1-mediated process. Our binding data also revealed that all tissue inhibitors of metalloproteases (TIMPs) bind to LRP1 with KD values ranging from 23 to 33 nM. Interestingly, the MMP-1/TIMP-1 complex bound to LRP1 with an affinity (KD = 0.6 nM) that was 30-fold higher than that of either component alone, revealing that LRP1 prefers the protease:inhibitor complex as a ligand. Of note, modification of lysine residues on either proMMP-1 or TIMP-1 ablated the ability of the MMP-1/TIMP-1 complex to bind to LRP1. LRP1's preferential binding to enzyme:inhibitor complexes was further supported by the higher binding affinity for proMMP-9/TIMP-1 complexes than for either of these two components alone. LRP1 has four clusters of ligand-binding repeats, and MMP-1, TIMP-1, and MMP-1/TIMP-1 complexes bound to cluster III most avidly. Our results reveal an important role for LRP1 in controlling ECM homeostasis by regulating MMP-1 and MMP-9 levels.

Itch and Nedd4 are members of the Nedd4 family of E3 ubiquitin ligases that are important in a number of biological processes. Precise regulation of their enzymatic activity is required for normal physiological function. Nedd4-like E3 ligases exist in an inactive form resulting from intramolecular interactions of their catalytic HECT domain with their WW domains. We identified the low-density-lipoprotein receptor class A domain containing 3 (LRAD3), a member of the LDL receptor family, as a potent activator of Itch and Nedd4 as evidenced by their increased auto-ubiquitination when bound to LRAD3. LRAD3 contains two PPxY motifs within its intracellular domain, both of which can bind to the WW domains on Itch and other Nedd4 family members with high affinity. Mutational analysis revealed that binding of Itch to the terminal LRAD3 PPxY motif is required to promote its auto-ubiquitination. We also determined that association of Itch and Nedd4 with LRAD3 leads to increased auto-ubiquitination and subsequent degradation through proteasome-mediated processes. Our findings reveal that LRAD3 is a component of pathways that function effectively to modulate Itch and Nedd4 auto-ubiquitination and levels. The identification of potential ligands for LRAD3 that may modulate LRAD3-induced activation of Itch and Nedd4 is likely to identify additional novel substrates and cellular functions for these important E3 ligases.

The reaction of several plasmin derivative with ..cap alpha../sub 2/-macroglobulin (..cap alpha../sub 2/M) has been investigated. Titration experiments measuring conformational changes in ..cap alpha../sub 2/M, changes in the number of sulfhydryl groups available for titration with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), and changes in the ability of ..cap alpha../sub 2/M to protect bound plasmin from inhibition by soybean trypsin inhibitor all suggested that between 1.3 and 1.5 mol of plasmin was bound per mole of inhibitor. Under experimental conditions where (plasmin) > (..cap alpha../sub 2/M), the conformational change occurring in the inhibitor and thiol group appearance displayed biphasic kinetics. Examination of the extent of subunit cleavage by plasmin revealed that the rapid phase was associated with cleavage of approximately two to three of the four ..cap alpha../sub 2/M subunits, while cleavage of the remaining subunits occurred during the slow phase of the reaction. Binary (1:1) ..cap alpha../sub 2/M-(/sup 125/I)-plasmin complexes were prepared. Characterization of the purified complex revealed that two of the four subunits were cleaved, and the conformational change,measured by alterations in the fluorescence of 6-(p-toluidino)-2-naphthalenesulfonate (TNS), was approximately 50% of that measured for a 2:1 complex. The association rate of Lys/sub 77/- and Val/sub 442/- plasmin with ..cap alpha../sub 2/Mmore » was examined under conditions where the concentration of ..cap alpha../sub 2/M exceeded that of plasmin, assuring that 1:1 complexes constitute the major product of the reaction. One millimolar element of-aminocaproic acid had no effect on the association rate while the presence of 12 ..mu..M histidine-rich glycoprotein (HRG) slightly reduced the association rate to 0.31 x 10/sup 5/ M/sup -1/ s/sup -1/. However, these molecules greatly reduce the association of plasmin with ..cap alpha../sub 2/-plasmin inhibitor lowering the rate to a value comparable to that measured for the reaction of plasmin with ..cap alpha../sub 2/M.« less