Abstract
The a disintegrin and metalloproteases (ADAMs) play a pivotal role in the control of development, adhesion, migration, inflammation and cancer. Although numerous substrates of ADAM10 have been identified, the regulation of its surface expression and proteolytic activity is still poorly defined. One current hypothesis is that both processes are in part modulated by protein-protein interactions mediated by the intracellular portion of the protease. For related proteases, especially proline-rich regions serving as docking sites for Src homology domain 3 (SH3) domain-containing proteins proved to be important for mediating regulatory interactions. In order to identify ADAM10-binding SH3 domain proteins, we screened the All SH3 Domain Phager library comprising 305 human SH3 domains using a GST fusion protein with the intracellular region of human ADAM10 as a bait for selection. Of a total of 291 analyzed phage clones, we found 38 SH3 domains that were precipitated with the ADAM10-derived fusion protein but not with GST. We verified the binding to the cytosolic portion of ADAM10 for several candidates by co-immunoprecipitation and/or pull down analyses. Intriguingly, several of the identified proteins have been implicated in regulating surface appearance and/or proteolytic activity of related ADAMs. Thus, it seems likely that they also play a role in ADAM10 biology.
Highlights
The a disintegrin and metalloprotease (ADAM) proteins form a subgroup of the metzincin superfamily that comprises matrix metalloproteinases (MMPs) and the a disintegrin and metalloproteases (ADAMs) proteases with thrombospondin motifs (ADAMTSs)
The screening was done in two separate experiments and revealed that out of the 305 human Src homology domain 3 (SH3) domains represented in the used phage library, as many as 38 were precipitated using the GSThADAM10(697–748) fusion protein (Table 1)
We focused on the SH3 domain proteins that did not precipitate with glutathione S-transferase (GST)
Summary
The a disintegrin and metalloprotease (ADAM) proteins form a subgroup of the metzincin superfamily that comprises matrix metalloproteinases (MMPs) and the ADAM proteases with thrombospondin motifs (ADAMTSs). ADAMs are glycosylated type I transmembrane proteins that are specialized for juxtamembrane cleavage of spatially associated membrane proteins [1,2]. This ectodomain shedding results in the release of bioactive extracellular protein fragments. ADAMs contain several distinct subdomains including a signal peptide, a pro-domain that is cleaved off during maturation, a metalloprotease domain, a disintegrin domain, a cysteine-rich region, an EGF (epidermal growth factor)-like or membrane-proximal domain, a transmembrane domain and an intracellular region (Fig. 1). The disintegrin domains guide interactions with integrins and the cysteine-rich domains support cell adhesion by binding to syndecans or fibronectin or clustering with other ADAMs [7]
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