ABSTRACT
The Slit-Robo GTPase-activating protein 3 (srGAP3) has been implicated in different critical aspects of neuronal development. These findings have mainly been based on the characterisation of the three conserved globular N-terminal domains, while the function of the C-terminal region (CTR) is still unknown. We show that this predicted unstructured region acts as an adaptor by binding to the endocytic proteins Amphiphysin, Endophilin-A2, Endophilin-A1, as well as the Ras signalling protein Grb2. All these interactions depend on a single proline-rich motif in the CTR and the Src-homology 3 domains of the binding partners. Via these interactions srGAP3 could link receptor signalling events to the endocytic machinery.
Subject(s)
Endocytosis/physiology , GTPase-Activating Proteins/metabolism , Signal Transduction/physiology , src Homology Domains , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Motifs , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/metabolism , GTPase-Activating Proteins/genetics , HEK293 Cells , Humans , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Protein BindingABSTRACT
Intersectin 1L (ITSN1L) acts as a specific guanine nucleotide exchange factor (GEF) for the small guanine nucleotide binding protein Cdc42 via its C-terminal DH domain. Interestingly, constructs of ITSN1L that comprise additional domains, for instance the five SH3 domains amino-terminal of the DH domain, were shown to be inhibited in their exchange factor activity. Here, we investigate the inhibitory mechanism of ITSN1L in detail and identify a novel short amino acid motif which mediates autoinhibition. We found this motif to be located in the linker region between the SH3 domains and the DH domain, and we show that within this motif W1221 acts as key residue in establishing the inhibitory interaction. This assigns ITSN1L to a growing class of GEFs that are regulated by a short amino acid motif inhibiting GEF activity by an intramolecular interaction. Moreover, we quantify the interaction between the ITSN1L SH3 domains and the Cdc42 effector N-WASP using fluorescence anisotropy binding experiments. As the SH3 domains are not involved in autoinhibition, binding of N-WASP does not release inhibition of nucleotide exchange activity in kinetic experiments, in contrast to earlier observations.
