Specific Interaction of the Recombinant Disintegrin-like Domain of MDC-15 (Metargidin, ADAM-15) with Integrin αvβ3

Abstract

MDC-15 (ADAM-15, metargidin), a membrane-anchored metalloprotease/disintegrin/cysteine-rich protein, is expressed on the surface of a wide range of cells and has an RGD tripeptide in its disintegrin-like domain. MDC-15 is potentially involved in cell-cell interactions through its interaction with integrins. We expressed a recombinant MDC-15 disintegrin-like domain as a fusion protein with glutathione S-transferase (designated D-15) in bacteria and examined its binding function to integrins using mammalian cells expressing different recombinant integrins. We found that D-15 specifically interacts with ␣v␤3 but not with the other integrins tested (␣2␤1, ␣3␤1, ␣4␤1, ␣5␤1, ␣6␤1, ␣6␤4, ␣v␤1, ␣IIb␤3, and ␣L␤2). Mutation of the tripeptide RGD to SGA totally blocked binding of D-15 to ␣v␤3, suggesting that D-15-␣v␤3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is recognized by both ␣IIb␤3 and ␣v␤3, suggesting that the receptor binding specificity is mediated by the sequence flanking the RGD tripeptide, as in snake venom disintegrins. These results indicate that the disintegrin-like domain of MDC-15 functions as an adhesion molecule and may be involved n ␣v␤3-mediated cell-cell interactions.Metalloprotease/disintegrin/cysteine-rich proteins (MDCs, also called ADAMs) 1 are membrane-anchored proteins with several domains including a metalloprotease domain, a disintegrin-like domain, a cysteine-rich sequence, an epidermal growth factor-like sequence, a transmembrane domain, and a short cytoplasmic domain (1). The biological functions of MDCs are not clear; however, we do know that fertilins (MDC-1 and -2) (2) are involved in sperm-egg binding and fusion (3), meltrins (MDC-12) (4) are involved in myoblast fusion during muscle development, and KUZ (a Drosophila MDC protein) (5) assists in neurogenesis. The MDC cytoplasmic domain has a proline-rich potential SH3 binding motif, suggesting that MDCcounter receptor interaction may induce signal transduction.Integrins are a family of cell adhesion receptors that bind to a variety of ligands, including extracellular matrix proteins and other cell surface molecules (6 -10). MDCs are potential ligands for integrins, since most snake venom disintegrins interact with integrins ␣IIb␤3 and ␣v␤3 (reviewed in Ref. 11 and references therein). However, little is known about the receptor specificity of MDCs, except that mouse egg integrin ␣6␤1 has been proposed as a receptor for fertilin (2). Evans et al. (12) recently expressed recombinant fertilin fragments in bacteria as fusion proteins with maltose-binding protein (12). The recombinant fertilin-␤ fragment has been shown to bind to the egg membrane to which sperm bind and to block sperm from binding to the egg. These results suggest that the disintegrinlike domains of MDCs may be properly folded in bacteria, that glycosylation of the disintegrin-like domain may not be required for interaction with receptors, and that a strategy using recombinant MDC proteins is a viable alternative to those using puri...

Distinct Functions of Natural ADAM-15 Cytoplasmic Domain Variants in Human Mammary Carcinoma

“…There is substantial evidence that ADAM-15 can influence both cell adhesion and migration (31,36,37). Interactions of ADAM-15 disintegrin domain with integrins a v h 3 , a 5 h 1 , and a 9 h 1 have been documented (5)(6)(7), and these may in principle occur either in trans, regulating cell-cell adhesion, or in cis, thereby modulating cell interactions with matrix substrata. Overexpression of ADAM-15 (this is likely the originally described ADAM-15A variant) in NIH 3T3 cells enhanced cell attachment and led to increased cell-cell adhesion, with a concomitant decrease in migration (37).…”

“…Human ADAM-15 is the only member of the ADAM family with the integrin-binding motif Arg-Gly-Asp (RGD) in its disintegrin domain (4). It binds to a v h 3 in monocyte-like U937 and melanoma cells and to a 5 h 1 in T-lymphocyte MOLT cells in an RGD-dependent manner (5,6) and also interacts with a 9 h 1 in an RGD-independent manner (7), suggesting that ADAM-15 may play a role in cellto-cell interactions and cell adhesion. The metalloproteinase domain of ADAM-15 has the potential for catalytic activity but its physiologic substrates are as yet undefined, although it was recently shown to cleave and activate heparin-binding epidermal growth factor in mammary cancer cells in response to thrombin (8).…”

Emerging roles of ADAM and ADAMTS metalloproteinases in cancer

“…Adhesion and migration of cells might be regulated by the disintegrin or cystein-rich domains whose importance has been evidenced by different studies [12,[61][62][63].…”

“…The different domains composing ADAMs have independent but complementary functions endowing these proteins with features of proteinases [11] and adhesion molecules [12] (Fig. 1).…”

“…It is worth noting that some ADAMs display alterations in this sequence resulting in a loss of proteolytic activity [14]. Although, the disintegrin domain has been widely described as being able to interact with integrin molecules and therefore mediating cell-cell and cell-matrix interactions [12,15,16], caution should be advised regarding this feature. In a recent study by Takeda et al, the disintegrin domain has been shown not to be available for protein binding due to protein folding [17].…”