SH3 Domain-dependent Association of Huntingtin with Epidermal Growth Factor Receptor Signaling Complexes

Abstract

Based on the presence of multiple proline-rich motifs in the huntingtin sequence, we tested its possible association with epidermal growth factor (EGF) receptor signaling complexes through SH3 domain-containing modules. We found that huntingtin is associated with Grb2, RasGAP, and tyrosine-phosphorylated EGF receptor. These associations are regulated by activation of the EGF receptor, suggesting that they may be part of EGF receptor-mediated cellular signaling cascade. In vitro binding studies indicate that SH3 domains of Grb2 or RasGAP are required for their binding to huntingtin. Our results suggest that huntingtin may be a unique adapter protein for EGF receptor-mediated signaling and may be involved in the regulation of Ras-dependent signaling pathways.Huntington's disease (HD) 1 is an autosomal dominant inherited neurodegenerative disorder characterized by massive loss of striatal neurons in caudate and putamen (1, 2). The defect in the HD gene locus involves a moderate expansion of the polymorphic CAG trinucleotide repeat near the N terminus of huntingtin (3). The age of onset of HD is roughly correlated to the number of the CAG repeats (4, 5). Although two proteins have been recently identified to interact with huntingtin in vitro (6, 7), the normal function of huntingtin remains a mystery.Identification of associated proteins is an initial step to characterize the biological function of any novel protein. Huntingtin does not contain any kinase, SH2, SH3, PH, or PTB domains, but does possess multiple proline-rich motifs in its sequence which resemble SH3 domain binding motifs (8,9). Most SH3 domain-containing proteins are adapter proteins for tyrosine kinase receptor-mediated signaling (8). We, therefore, examined the possible association of huntingtin with several SH3 domain-containing signaling molecules in EGF receptor signaling complexes. We found that huntingtin is directly associated with both Grb2 and RasGAP and indirectly associated with Shc and the EGF receptor. Huntingtin is not associated with Nck and does not co-exist with Sos in the same signaling complex. Our results suggest that huntingtin may be a unique signaling intermediate for EGF receptors and may be involved in the regulation of Ras-dependent signaling pathways. EXPERIMENTAL PROCEDURESImmunoblotting and Co-immunoprecipitation-Human epithelial carcinoma A431 or embryonic kidney 293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. These cells, starved in serum-free medium for 24 -48 h, were stimulated with 100 ng/ml EGF for 5 min at 37°C. Cells were then washed once with ice-cold phosphate-buffered saline (PBS) and lysed with Nonidet P-40 lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 1% Nonidet P-40, 1 mM sodium fluoride, 1 mM sodium orthovanadate, 10% glycerol, 100 M phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin, 10 g/ml pepstatin A). The antihuntingtin monoclonal antibody 4C8 has been characterized previously (10). The anti-huntingtin pol...

Mutant huntingtin can paradoxically protect neurons from death

“…[29][30][31][32][33] Other candidates that possibly could interact with the domain of the protein encoded by exon 1, but have not been shown to interact only with this short sequence, include huntingtin interacting protein 1 (HIP1), Rab11 familyinteracting protein 2 (FIP2), huntingtin interacting protein 14 (HIP14), nuclear receptor corepressor 1 (N-CoR) and cystathionine beta-synthase (CBS) [34][35][36][37][38] (for overview see Figure 2). …”

“…It is not known if the interaction between GRB2 and Huntingtin is dependent on the poly-Q length of Huntingtin. 30 A GRB2-like protein, SH3GL3 (endophilin 3), also binds to exon 1 Huntingtin. This interaction is discussed as a regulating factor for dynamin and synaptojanin function.…”

C. elegans comme modèle pour les maladies dégénératives héréditaires humaines

“…En particulier, les phénotypes d'inactivation du gène codant pour la huntingtine chez la souris suggèrent que la protéine serait impliquée dans la neurogenèse [19] et dans la survie neuronale à l'âge adulte [20]. Les propriétés toxiques induites par la huntingtine mutée et les « pertes de fonction » résultant de l'absence de la protéine normale sont d'une grande diversité: anomalies du trafic et de la dégradation des protéines, anomalies de la transduction du signal [24][25][26] et anomalies de la transcription [27] provoquant éventuellement des pertes d'expression de neurotrophines comme le BDNF (brain-derived neurotrophic factor) [28] ou le NGF (nerve growth factor) [29]. Certains aspects de la maladie restent cependant sujet à controverse, comme par exemple l'éventuelle toxicité des agrégats, structures hété-rogènes et réversibles [30], par rapport à celle des protéines mutantes solubles [31].…”

Cdc42-interacting protein 4 binds to huntingtin: Neuropathologic and biological evidence for a role in Huntington's disease

“…The htt-associated protein HAP1 binds to Duo, a Trio-like protein that contains a Rac1 guanine nucleotide exchange factor (GEF) domain, raising the possibility that polyQ-expanded htt may affect a ras-related signaling pathway (18). Normal htt is associated with the Cdc42 interactor Grb2, the Ras GTPaseactivating protein (RasGAP), and epidermal growth factor (EGF) receptor (19). Mutant htt disrupts cellular signaling mediated by the EGF receptor in PC12 cells (20).…”