Preferred SH3 Domain Partners of ADAM Metalloproteases Include Shared and ADAM-Specific SH3 Interactions

Abstract

A disintegrin and metalloproteinases (ADAMs) constitute a protein family essential for extracellular signaling and regulation of cell adhesion. Catalytic activity of ADAMs and their predicted potential for Src-homology 3 (SH3) domain binding show a strong correlation. Here we present a comprehensive characterization of SH3 binding capacity and preferences of the catalytically active ADAMs 8, 9, 10, 12, 15, 17, and 19. Our results revealed several novel interactions, and also confirmed many previously reported ones. Many of the identified SH3 interaction partners were shared by several ADAMs, whereas some were ADAM-specific. Most of the ADAM-interacting SH3 proteins were adapter proteins or kinases, typically associated with sorting and endocytosis. Novel SH3 interactions revealed in this study include TOCA1 and CIP4 as preferred partners of ADAM8, and RIMBP1 as a partner of ADAM19. Our results suggest that common as well as distinct mechanisms are involved in regulation and execution of ADAM signaling, and provide a useful framework for addressing the pathways that connect ADAMs to normal and aberrant cell behavior.

The Cxadr–Adam10 complex plays pivotal roles in tight junction integrity and early trophoblast development in mice

“…ADAM10 is co‐localised at the AJ of the basolateral cell membrane in MDCK cells, and the intracellular regions of ADAM10 interact with SH3 domains of transmembrane proteins mediated via TJP1 (Kleino, Jarviluoma, Hepojoki, Huovila, & Saksela, ). TJP1 is involved in establishing the TJ complex by interacting with other components, such as OCLN or CXADR (Excoffon, Hruska‐Hageman, Klotz, Traver, & Zabner, ; Fanning, Jameson, Jesaitis, & Anderson, ).…”

Large-Scale Screening of Preferred Interactions of Human Src Homology-3 (SH3) Domains Using Native Target Proteins as Affinity Ligands

“…The phage library that we used has previously proven its value in identifying high-affinity SH3 partners for a number of cellular and pathogen-encoded ligand proteins (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25). These studies have established the positive predictive value of the hits generated by this screening method to be remarkably high, i.e.…”

“…Because these target proteins are expressed in their native form, this system has the potential to explore binding affinity and specificity contributed by contacts provided by the peptide binding interface, as well as more complex and atypical interactions. The binding affinity required for positive identification of a specific interaction in this discovery system is relatively high (estimated to be in the range of 2 to 5 M), as interactions with dissociation constants higher than 5 M are rarely found (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) and unpublished observations). Although this may be seen as a technical limitation when considering that many SH3 interactions with established roles in cell biology are weak such an affinity threshold is also a major experimental advantage by filtering out nonspecific background caused by promiscuous low affinity binding that most SH3 domains exhibit toward a variety of proline-rich sequences.…”

ANKRD54 preferentially selects Bruton’s Tyrosine Kinase (BTK) from a Human Src-Homology 3 (SH3) domain library

“…The pEBB-vector expresses a biotinylation target-domain (BTD) fusion, defined by a single lysine residue that serves as the biotin acceptor. The BTD exists as biotin acceptor domain of the 1.3S subunit of Propionibacterium shermanii originating from the PinPoint (pp) Xa-1 vector, as described in [35]. The ANKRD54 wild-type and ANKRD54-Δ3 sequences were sub-cloned into the C-terminal position of the pEBB backbone (pEBB-pp-ANKRD54), which was cleaved with BamHI and KpnI.…”

“…SH3 phage libraries were prepared as described in [25] and the bio-panning procedure to select human SH3-domains binding to ANKRD54 was carried out according to [25] and [35]. Briefly, the biotinylated fusion protein of interest, ANKRD54 wild-type was analyzed with the ANKRD54-Δ3 mutant as a negative control and human immunodeficiency virus-1 (HIV Nef) as positive control, following transient transfection into HEK 293T cells.…”