Cytosolic SH3-interactions of ADAM12-disintegrin-metalloprotease
TEITTINEN, KAISA (2007)
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TEITTINEN, KAISA
2007
Biokemia - Biochemistry
Lääketieteellinen tiedekunta - Faculty of Medicine
Hyväksymispäivämäärä
2007-05-15Tiivistelmä
Background: ADAM proteins (a disintegrin and metalloprotease) are membrane proteins that consist of several functional domains. ADAM proteins are able to mediate cell adhesion through their disintegrin domain and selective protein cleavage (e.g. ectodomain shedding) carried out by the metalloprotease domain. ADAM12 is an important sheddase, expressed widely in different tissues. ADAM12 has been associated with e.g. regulation of muscle and adipose tissue differentiation, cell fusion, cell adhesion, and EGFR-ligand activation. ADAM12 cytosolic tail contains several recognition motifs for adaptor and signaling proteins and interactions with such proteins are thought to regulate the function of ADAM12. In this study the focus was on ADAM12-SH3 interactions and the main objective was to identify the SH3 proteins that bind to human ADAM12 cytosolic tails and to further characterize these binding interactions.
Methods: Human ADAM12 cytosolic tail was expressed as recombinant GST-fusion protein. The SH3 domains that can bind to ADAM12 cytosolic tail were identified using phage display method and selected individual ADAM12-SH3 interactions as well as the SH3-binding motifs of the ADAM12 cytosolic tail were further characterized in peptide array.
Results: The screening of SH3-phage display library revealed altogether 50 SH3 proteins that bound to human ADAM12 cytosolic tail. The phage display and peptide array screens showed that ADAM12 cytosolic tail motifs differed in their SH3-binding specificity. The peptide screen results indicate that the proline cluster P3 mediated more SH3-interactions than other proline clusters in the ADAM12 cytosolic tail.
Conclusions: The proline-rich regions of ADAM12 cytosolic tail clearly mediate SH3-interactions. The large number of identified interacting SH3 proteins can be seen as an indicator of potential importance of ADAM12 in different physiological processes such as signaling pathways as an active mediator or in a more structural role. Altogether, identification of candidate regulatory interactions provides important clues for future studies addressing the functional regulation of ADAM12.
Asiasanat: ADAM12-disintegrin-metalloprotease, SH3 domain interaction, phage display, peptide array
Methods: Human ADAM12 cytosolic tail was expressed as recombinant GST-fusion protein. The SH3 domains that can bind to ADAM12 cytosolic tail were identified using phage display method and selected individual ADAM12-SH3 interactions as well as the SH3-binding motifs of the ADAM12 cytosolic tail were further characterized in peptide array.
Results: The screening of SH3-phage display library revealed altogether 50 SH3 proteins that bound to human ADAM12 cytosolic tail. The phage display and peptide array screens showed that ADAM12 cytosolic tail motifs differed in their SH3-binding specificity. The peptide screen results indicate that the proline cluster P3 mediated more SH3-interactions than other proline clusters in the ADAM12 cytosolic tail.
Conclusions: The proline-rich regions of ADAM12 cytosolic tail clearly mediate SH3-interactions. The large number of identified interacting SH3 proteins can be seen as an indicator of potential importance of ADAM12 in different physiological processes such as signaling pathways as an active mediator or in a more structural role. Altogether, identification of candidate regulatory interactions provides important clues for future studies addressing the functional regulation of ADAM12.
Asiasanat: ADAM12-disintegrin-metalloprotease, SH3 domain interaction, phage display, peptide array