RESUMO
Integrin alpha2beta1 is the principal adhesive receptor for collagen but platelets also adhere through glycoprotein VI (GPVI). Integrin alphaIIbbeta3 may augment platelet adhesion. We have shown that disulfide exchange is necessary for platelet adhesion to fibrinogen, fibronectin, and collagen. However 2 questions remained: (1) Can activated alphaIIbbeta3 explain the observed role of disulfide exchange in adhesion to collagen, or is this role common to other integrins? (2) Is disulfide dependence specific to the integrin receptors or shared with GPVI? To discriminate adhesive functions of alpha2beta1 from those of alphaIIbbeta3 we used Glanzmann platelets and alphaIIbbeta3-specific antibodies applied to normal platelets. To resolve adhesive events mediated by alpha2beta1 from those of GPVI we used synthetic peptides specific to each receptor. We addressed direct integrin ligation using purified alpha2beta1 and recombinant I domain. We observed the following: adhesion to the alpha2beta1-specific peptide was disulfide-exchange dependent and protein disulfide isomerase (PDI) mediated; membrane-impermeant thiol blockers inhibited alpha2beta1, but not GPVI mediated, adhesion; direct blockade of PDI revealed that it is involved in adhesion through alpha2beta1 but not GPVI; and purified alpha2beta1, but not recombinant I domain, depended on free thiols for ligation. These data suggest that the enzymatically catalyzed adhesion-associated reorganization of disulfide bonds is common to members of the integrin family and specific to this family.
Assuntos
Colágeno/metabolismo , Dissulfetos/metabolismo , Integrina alfa2beta1/metabolismo , Adesividade Plaquetária/fisiologia , 4-Cloromercuriobenzenossulfonato/farmacologia , Antibacterianos/farmacologia , Anticorpos/farmacologia , Bacitracina/farmacologia , Proteínas de Transporte/metabolismo , Catálise , Reagentes de Ligações Cruzadas/metabolismo , Ácido Ditionitrobenzoico/farmacologia , Relação Dose-Resposta a Droga , Humanos , Integrina alfa2beta1/química , Ligantes , Peptídeos/metabolismo , Piperazinas/farmacologia , Piperidinas/farmacologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/imunologia , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Isomerases de Dissulfetos de Proteínas/antagonistas & inibidores , Isomerases de Dissulfetos de Proteínas/imunologia , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Compostos de Sulfidrila/metabolismo , Reagentes de Sulfidrila/farmacologiaRESUMO
To be able to understand cellular mechanisms, we require fully integrated data sets combining information about gene expression, protein expression, post-translational modification states, sub-cellular location and complex formation. Proteomics is a very powerful technique that can be applied to interrogate changes at the protein level. Studying this effectively requires specialised facilities within research institutes. Here, we describe the setting up and operation of such a facility, providing a resource for the Arabidopsis and Drosophila research communities.
