On the Function of Variable Regions in the F-BAR Proteins SrGAP3 and Syndapin1

Abstract

Cellular signalling and membrane trafficking critically depend on the assembly of proteins into large complexes via transient protein-protein and protein-lipid interactions. For this purpose, involved proteins possess different adaptor domains, which are combined based on a modular principle in a single polypeptide chain. A prevalent interaction mode constitutes binding of adaptor domains to linear motifs, which are frequently located in intrinsically disordered regions. The typically low affinity and promiscuous nature, with which linear motifs bind their targets, establishes reversibility and flexibility within the system. Additional membrane-interaction modules, such as F-BAR (Fes-Cip4-homology Bin-Amphiphysin-Rvs) domains, assist not only in the localisation of these multi-protein complexes, but can also affect membrane shape, which is a prerequisite for membrane trafficking. In this thesis, I have investigated linear motifs in the variable regions of two F-BAR domain-containing proteins, srGAP3 (Slit-Robo GTPase-activating protein 3) and Syndapin1 (synaptic Dynamin-associated protein 1), with respect to binding partners and functional implications of the formed complexes.

SrGAP proteins have been implicated in different aspects of neuronal development and were identified as downstream effectors in the Slit-Robo pathway, in which they transmit the signal to the actin cytoskeleton, thereby affecting targeting of axons and neuronal migration. These findings were mainly based on the characterisation of their highly conserved globular domains, while the function of the variable C-terminal region (CTR) remained elusive. My bioinformatic analysis predicts that this region of srGAP proteins is intrinsically disordered and contains linear motifs for the interaction with 14-3-3 proteins as well as Src-homology 3 (SH3) domains. 14-3-3 proteins typically engage in phospho-dependent interactions, while SH3 domains recognise proline-rich motifs. Employing different biochemical and biophysical methods, I show that the CTR of the family member srGAP3 confers binding to the SH3 domains of the endocytic proteins Endophilin-A1, Endophilin-A2, and Amphiphysin, as well as the Ras signalling adaptor Grb2 under in vivo conditions and in vitro. Moreover, a single proline-rich motif in the CTR is sufficient for all of these interactions. Furthermore, I provide evidence that the CTR binds the 14-3-3 isoforms gamma and theta/tau in vivo and I identify two phosphorylated positions involved in the formation of these complexes. These results therefore not only uncover a potential function of srGAP3 in endocytosis and Ras signalling, but also suggest that the CTR is subject to phosphoregulation and may act as an adaptor platform connecting srGAP3 with different protein networks.

Syndapins have likewise been implicated in endocytic processes, in which they function as auxiliary factors during vesicle formation. Vesicle trafficking requires the separation and fusion of membranes through a concerted action of membrane-bending proteins, the lipid composition, and the actin cytoskeleton, which have to be precisely regulated in time and space. The F-BAR domain of Syndapin1 exhibits potent membrane sculpting activity, which is regulated through an autoinhibitory clamp between the C-terminal SH3 domain and the N-terminal F-BAR domain and is activated upon binding of ligands to the SH3 domain. I show that binding of the Eps15-homology domain-containing protein 1 (EHD1) to two Asn-Pro-Phe motifs located in the central variable region of Syndapin1 constitutes an alternative route for release of the autoinhibition. The Syndapin1-EHD1 complex plays a role in endocytic receptor recycling and, therefore, I propose that activation of Syndapin1 by EHD1 is a crucial step in the molecular mechanism underlying membrane trafficking processes governed by this protein complex.