Specificity of adaptor proteins involved in signal transduction pathways and endocytosis

Group Leader: Dr. Yvonne Groemping

MPI for Developmental Biology
Department of Protein Evolution
Spemannstr. 35
72076 Tübingen, Germany

Phone: +49 - 7071 - 601 356
email: yvonne.groemping [at] tuebingen.mpg.de

Group members

Open positions

The lab has an open position for a Postdoctoral fellow. We are searching for commited, enthusiastic individuals with a background in biomolecular science. The applicant should have a PhD in biology, biochemistry, chemistry or related subject. Experience in quantification of protein-protein interaction or structural biology would be an advantage. To apply, please send an email including a cover letter and a CV to: yvonne.groemping [at ]tuebingen.mpg.de.

Publications

Research interests

The process of transduction of an extracellular signal from the plasma membrane to specific sites inside the cell requires the controlled formation of multi protein networks. Adaptor and scaffold proteins play an important role in facilitating protein-protein interactions and thus the formation of these networks. Adaptor proteins can recruit binding partners to a specific location and regulate interactions between different signal transduction proteins. Crucial for the formation of such networks are reversible protein-protein interactions. Adaptor proteins usually consist of different protein-protein and protein-lipid interaction modules like the SH (src homology) 2 and 3 domains that bind a short target sequence within the binding partner. Lipid-interaction modules like the PH (pleckstrin homology) or PX (phox homology) domains bind phosphatidylinositides and determine the localisation of the adaptor protein.

By this means adaptor proteins can amplify a receptor-mediated signal and facilitate coupling of the signal to different signal transduction pathways.

We are interested in how these adaptor proteins function to interact with different binding partners in the process of signal transduction and how they are regulated. We use a wide range of methods such as molecular biology, fluorescence spectroscopy, microcalorimetry and structural techniques. Aim of our research is to understand the coordination of different signal transduction pathways and principles of substrate recognition and specificity of adaptor proteins.
This project is funded by an Emmy-Noether fellowship from the DFG and the Max Planck Gesellschaft.

Intersectin

The multi domain adaptor protein Intersectin is involved in several signal transduction pathways in the cell. It interacts with various components of the endocytic pathway and plays an important role in regulation of Ras induced activation of MAPK kinases and other GTPase regulated processes. The human gene for Intersectin 1 is located in the Down Syndrome region of chromosome 21 (Pucharcos et al., 1999).


Intersectin - at the intersection of major cellular signalling pathways

Intersectin combines different protein-protein interaction modules. At the N-terminus Intersectin exhibits two Eps15 homology domains that are found in proteins of the endocytic pathway. EH domains are evolutionary conserved protein interaction domains with a number of cellular ligands. Via these domains it binds a specific motif- the NPF (asparagin, proline, phenylalanin)-motif in other endocytic proteins like Epsin or Synaptojanin (Santolini et al., 1999). The coiled-coil domain of Intersectin is supposed to be responsible for the formation of homodimers and heterodimers with Eps15 (Santolini et al., 1999),(Sengar et al., 1999).

Intersectin contains 5 SH3-domains. These domains mediate protein-protein interaction by binding specific proline-rich stretches within other proteins. Using immunoprecipitation assays and GST-pulldown experiments i.a. many binding partners for these SH3 domains were identified (Yamabhai et al., 1998), (Sengar et al., 1999), (Roos and Kelly, 1998), (Tong et al., 2000), (Adams et al., 2000). Among them are endocytic proteins like dynamin or synaptojanin and proteins involved in other signalling pathways such as mSos, an exchange factor for Ras and N-WASP, a regulator of actin polymerisation.

A longer splicing variant of Intersectin additionally carries a Dbl homology (DH), Pleckstrin homology (PH) and C2-domain. DH domains catalyse the nucleotide exchange for the family of Rho GTPases and are usually found adjacent to PH domains, which bind phospholipids and thus could be responsible for membrane anchoring of the exchange factors.


Domain structure of the adaptor protein Intersectin

Intersectin as nucleotide exchange factor for Cdc42

Eps15: A high affinity EH domain complex

Eps15 homology (EH) domains are important mediators of protein-protein interactions in various trafficking processes in the cell. EH domains were shown to bind motifs containing the amino acid residues Asn-Pro-Phe (NPF) in unfolded regions of their binding partners (de Beer et al., 1998; Confalonieri and Di Fiore, 2002; Morgan et al., 2003). So far, interactions between EH-domains and their respective ligands were reported to be of low affinity and also low specificity (Salcini et al., 1997).


We discovered a highly specific, high affinity interaction between Eps15, an essential accessory protein during clathrin mediated endocytosis, and its binding partner Stonin2 (see Rumpf et al., 2008). Stonin2 was shown to act as a specific sorting adaptor for the internalisation of synaptotagmin and thus to be a regulator of synaptic vesicle recycling (Diril et al., 2006).


Eps15 is a multi-modular protein that comprises 3 EH domains, a coiled-coil region and a low complexity region at its c-terminus that harbours several peptide motifs, shown to interact with other endocytic proteins, for example the AP-2 complex (Figure 1). Stonin2 consists of a ? homology domain, a Stonin homology (SH) domain and a low complexity N-terminus with 2 NPF motifs.




Eps15-EH1-3 binds Stonin2 NPF-region with an apparent affinity of 0.15 uM, about three orders of magnitude tighter than observed for other EH-NPF complexes.
This high-affinity interaction is mediated by the second EH domain of Eps15 only (Figure 2). EH1 binds with low affinity to the Stonin2 NPF region, while we cannot observe a heat change with EH3 in the calorimeter. On the other hand, both NPF motifs of Stonin2 are required for a tight binding. Mutations of NPF1 or 2 to triple alanines either significantly reduce the affinity (NPF2-AAA) or complete abrogate the interaction (NPF1-AAA).

 


  We solved the structure of the complex consisting of Eps15-EH2 and the Stonin2 NPF-region in collaboration with Michael Sattler’s group, using NMR spectroscopy (see also (Rumpf et al., 2008a). The structure (Figure 4) shows how both NPF motifs can simultaneously interact with one EH domain. Whereas NPF1 binds into the conserved ligand binding pocket between helices 2 and 3, the second NPF motif inserts into a groove formed by helices 1, 2 and 4. This newly identified pocket is far less conserved and the critical residues that mediate interaction to NPF2 are only present in Eps15 and Eps15R-EH2 domains.
The first binding site resembles previously characterized EH-NPF complexes (de Beer et al., 2000). Only few contacts were observed for residues outside the core NPF motif. Thus, this binding site is more promiscuous when it comes to ligand binding and would interact with a number of ligands with relatively low affinity. In contrast, the second binding site is characterized by a high number of contacts between residues flanking NPF2 and the EH domain. In particular the hydrophobic residues preceding NPF2 (Pro326 and Ile327) as well as two Phe residues (334-335) following the motif are involved in the interaction. These structural observations could be confirmed by a detailed study using binding site mutants, see (Rumpf et al., 2008b). Although this newly identified binding pocket is unique among human EH domains, it is conserved regarding Eps15 proteins throughout evolution.

Kintscher, C and Groemping, Y (2009). Characterisation of the nucleotide exchange factor ITSN1L: Evidence for a kinetic discrimination of GEF stimulated nucleotide release from Cdc42. J Mol Biol 387: 270-283.

Rumpf J, Simon B, Jung N, Maritzen T, Haucke V, Sattler M, Groemping Y (2008). Structure of the Eps15-stonin2 complex provides a molecular explanation for EH domain ligand specificity. EMBO J 27: 558-569.

Groemping Y and Rittinger K (2005). Activation and assembly of the NADPH oxidase: a structural perspective. Biochem J 386: 401-416.

Groemping Y, Lapouge K, Smerdon SJ and Rittinger K (2003). Molecular basis of phosphorylation-induced activation of the NADPH oxidase. Cell 113: 343-55.

Groemping Y and Reinstein J (2001). Folding properties of the nucleotide exchange factor GrpE from Thermus thermophilus: GrpE is a thermosensor that mediates heat shock response. J Mol Biol 314: 167-78.

Groemping Y, Klostermeier D, Herrmann C, Veit T, Seidel R, Reinstein J (2001). Regulation of ATPase and chaperone cycle of DnaK from Thermus thermophilus by the nucleotide exchange factor GrpE. J Mol Biol 305: 1173-83.