Three Proteins Regulating Integrin Function – Filamin, 14-3-3 and RIAM

Takala, Heikki Three proteins regulating integrin function – filamin, 14-3-3 and RIAM Jyväskylä: University of Jyväskylä, 2011, 78 p. (Jyväskylä Studies in Biological and Environmental Science ISSN 1456-9701; 232) ISBN 978-951-39-4523-7 (nid.) ISBN 978-951-39-4524-4 (PDF) Yhteenveto: Kolme integriinin toimintaa säätelevää proteiinia – filamiini, 14-3-3 ja RIAM Diss. Integrins are transmembrane adhesion receptors important in cell adhesion, migration and survival. Integrin activation is regulated by cytoplasmic protein– protein interactions. Talin activates integrins by binding to integrin cytoplasmic tails. As dozens of other cytoplasmic proteins interact with integrins, their mutual interactions and function in integrin cytoplasmic adhesion structures have to be carefully regulated. This is achieved, e.g., by protein recruitment, competitive binding, alternative splicing and protein phosphorylation. The Rap1-GTP interacting adapter molecule (RIAM) recruits talin to integrins with Rap1 GTPase, thereby facilitating integrin activation. Filamins compete with talin for integrin binding. This is expected to be particularly important for a splice variant-1 (var-1) of filamin A (FLNa) that lacks a segment with an autoinhibitory role in integrin binding. The cytoplasmic tail of 2 integrin is phosphorylated on a threonine residue upon inside-out activation events, which is accompanied by 14-3-3 binding. In this thesis, the structural and functional properties of RIAM, FLNa var-1 and 14-3-3 were studied. RIAM contains a Ras-association (RA) domain followed by a pleckstrin homology (PH) domain. We showed that the RA domain is responsible for the binding to Rap1, whereas the PH domain accounts for the stability of the protein. FLNa var-1 lacks a segment of 41 residues. We showed that this absence causes domain 19 to be unstructured and unable to bind ligands, but it does not seem to significantly affect the binding properties of domain 21. The crystal structure of 14-3-3 in complex with the phosphorylated 2 integrin peptide reveals at the atomic level how this interaction is dependent on the phosphate moiety. The same phosphorylation abrogates FLNa binding but does not affect the binding of talin. However, talin binding to the phosphopeptide is outcompeted by 14-33 , which is explained by the partially overlapping binding sites on the integrin.