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Specific interaction of the recombinant disintegrin-like domain of MDC-15 (metargidin, ADAM-15) with integrin
X P Zhang1, T Kamata, K Yokoyama
1Department of Vascular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
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 alphavbeta3 but not with the other integrins tested (alpha2beta1, alpha3beta1, alpha4beta1, alpha5beta1, alpha6beta1, alpha6beta4, alphavbeta1, alphaIIbbeta3, and alphaLbeta2). Mutation of the tripeptide RGD to SGA totally blocked binding of D-15 to alphavbeta3, suggesting that D-15-alphavbeta3 interaction is RGD-dependent. When the sequence RPTRGD is mutated to NWKRGD, D-15 is recognized by both alphaIIbbeta3 and alphavbeta3, 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 alphavbeta3-mediated cell-cell interactions.
Insights
Metargidin (MDC-15) disintegrin-like domain specifically binds to integrin alphavbeta3. This interaction, mediated by the RGD motif, suggests MDC-15
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- MDC-15 (metargidin) is a metalloprotease with a disintegrin-like domain, expressed on cell surfaces.
- It contains an RGD tripeptide motif, suggesting potential interactions with integrins.
- Integrins are cell surface receptors involved in cell adhesion and signaling.
Purpose of the Study:
- To investigate the binding specificity of the MDC-15 disintegrin-like domain to various integrins.
- To determine the role of the RGD motif and flanking sequences in MDC-15-integrin interactions.
Main Methods:
- Expression of a recombinant MDC-15 disintegrin-like domain (D-15) as a fusion protein.
- Testing D-15 binding to mammalian cells expressing different recombinant integrins.
- Site-directed mutagenesis of the RGD motif and flanking sequences in D-15.
Main Results:
- D-15 specifically bound to integrin alphavbeta3, but not other tested integrins.
- Mutation of the RGD to SGA abolished D-15 binding to alphavbeta3, confirming RGD-dependent interaction.
- Altering flanking sequences (RPTRGD to NWKRGD) enabled binding to both alphavbeta3 and alphaIIbbeta3.
Conclusions:
- The disintegrin-like domain of MDC-15 functions as an adhesion molecule.
- MDC-15 specifically interacts with integrin alphavbeta3 in an RGD-dependent manner.
- Flanking sequences modulate the receptor binding specificity of MDC-15, similar to snake venom disintegrins.