RIN3: a novel Rab5 GEF interacting with amphiphysin II involved in the early endocytic pathway

Background:The CD2AP adaptor facilitates cell signaling using its in-built interaction modules (SH3 domains). Results: RIN3 was characterized as a novel CD2AP SH3 binding protein by biophysical and biochemical methods. Conclusion: CD2AP has many potential interactors and is probably a cellular hub. Significance: We reveal how protein modules can interact with families of related recognition motifs rather than a single motif.

Section: Discussionmentioning

confidence: 99%

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confidence: 99%

Differential Recognition Preferences of the Three Src Homology 3 (SH3) Domains from the Adaptor CD2-associated Protein (CD2AP) and Direct Association with Ras and Rab Interactor 3 (RIN3)

Rouka

Simister

Janning

et al. 2015

“…Our previous work suggested that Rin1 is an exchange factor for small GTPase Rab5, whose overexpression stimulates EGF-mediated endocytosis (31). The Rin family now has at least four members, all of which have active Rab5 guanine nucleotide exchange factor domains (32)(33)(34)(35). Subsequent work showed that Rin1 is recruited to the EGFR via its SH2 domain and that it regulates EGF-induced signal transduction (36,37).…”

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confidence: 99%

Rin1 Interacts with Signal-transducing Adaptor Molecule (STAM) and Mediates Epidermal Growth Factor Receptor Trafficking and Degradation

Chen

et al. 2007

Rin1, the prototype of a new family of multidomain Rab5 exchange factors, has been shown to play an important role in the endocytosis of the epidermal growth factor receptor (EGFR). Herein, we examined the role of Rin1 in the down-regulation of EGFR following EGF stimulation. We observed that overexpression of Rin1 accelerates EGFR degradation in EGF-stimulated cells. In concordance, depletion of endogenous Rin1 by RNA interference resulted in a substantial reduction of EGFR degradation. We showed that Rin1 interacts with signal-transducing adaptor molecule 2 (STAM2), a protein that associates with hepatocyte growth factor-regulated substrate and plays a key role in the endosomal sorting machinery. Green fluorescent protein (GFP)-Rin1 co-localizes with hemagglutinin (HA)-STAM2 and with endogenous hepatocyte growth factor-regulated substrate. Furthermore, wild type STAM2, but not a deletion mutant lacking the SH3 domain, co-immunoprecipitates with endogenous Rin1. This interaction is dependent on the proline-rich domain (PRD) of Rin1 as Rin1⌬PRD, a mutant lacking the PRD, does not interact with STAM2. Moreover, EGFR degradation was not accelerated by expression of the Rin1⌬PRD mutant. Together these results suggest that Rin1 regulates EGFR degradation in cooperation with STAM, defining a novel role for Rin1 in regulating endosomal trafficking. The epidermal growth factor receptor (EGFR)2 plays a central role in cell proliferation, differentiation, survival, and migration (1, 2) and has served as a prototype in growth factor receptor trafficking. Following activation, EGFR with intrinsic tyrosine kinase activity is rapidly internalized by clathrincoated pits (3), sorted through early endosomes, and eventually transported to and degraded within multivesicular bodies (MVB) and lysosomes (4, 5). The process is generally known as receptor down-regulation and is considered to be an important cellular strategy for signal attenuation (6, 7). Alterations in receptor trafficking and signal attenuation have been associated with carcinogenesis (5, 8) and certain developmental processes, which has stimulated interest in the molecular mechanisms that regulate EGFR trafficking and degradation.EGFR targeted for lysosomal degradation is delivered to the MVB by a highly specialized process that begins with receptor ubiquitination and sequestration by elements of the ESCRT (endosomal sorting complex required for transport) complex on the surface of the early endosomes (9, 10). Invagination of the endosomal membrane and delivery of sequestered receptors into the lumen of the MVB is accompanied by receptor deubiquitination and disassembly of the ESCRT complex (11,12). The ESCRT machinery, first identified in yeast as class E Vps (vacuolar protein sorting) mutants (13) and highly conserved among eukaryotic cells (14), is important in targeting EGFR into the lumen of the MVB (15, 16). Substantial progress has been made in identifying the molecular mechanisms involved (12,17). Among the early acting factors are Hrs (hepatocyte growth fa...

“…RLD is followed by a tandem organization of Dbl homology (DH) and pleckstrin homology (PH) domains, which is a hallmark for GEFs for Rho (Ras homologous member) GTPases (14). The C-terminal end of ALS2 harbors a vacuolar protein sorting 9 (VPS9) domain, which has been found in Rab5 (Ras-related in brain 5) GEFs, including Vps9 (15), , RIN1 (17,18), RIN2 (19), and RIN3 (20). In addition, eight consecutive membrane occupation and recognition nexus (MORN) motifs (21) were noted in the region between PH and VPS9 domains.…”

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confidence: 99%

Homo-oligomerization of ALS2 through Its Unique Carboxyl-terminal Regions Is Essential for the ALS2-associated Rab5 Guanine Nucleotide Exchange Activity and Its Regulatory Function on Endosome Trafficking

Kunita

Otomo

Mizumura

et al. 2004

Mutations in the ALS2 gene have been known to account for a juvenile recessive form of amyotrophic lateral sclerosis (ALS2), a rare juvenile recessive form of primary lateral sclerosis, and a form of hereditary spastic paraplegia (HSP), indicating that the ALS2 protein is essential for the maintenance of motor neurons. Recently, we have demonstrated that the ALS2 protein specifically binds to the small GTPase Rab5 and acts as a GEF (guanine nucleotide exchange factor) for Rab5. We have also shown that its Rab5GEF-requisite domain resides within the C-terminal 640-amino acid region spanning membrane occupation and recognition nexus motifs and the vacuolar protein sorting 9 domain. Transiently expressed ALS2 localized onto early endosomal compartments and stimulated endosome fusions in neuronal and non-neuronal cells in an Rab5GEF activity-dependent manner. These results indicate that the C-terminal region of ALS2 plays a crucial role in endosomal dynamics by its Rab5GEF activity. Here we delineate a molecular feature of the ALS2-associated function through the C-terminal region-mediated homo-oligomerization. A yeast two-hybrid screen for interacting proteins with the ALS2 C-terminal portion identified ALS2 itself. ALS2 forms a homophilic oligomer through its distinct C-terminal regions. This homo-oligomerization is crucial for the Rab5GEF activity in vitro and the ALS2-mediated endosome enlargement in the cells. Taken together, these results indicate that oligomerization of the ALS2 protein is one of the fundamental features for its physiological function involving endosome dynamics in vivo.ALS2 was initially identified as a causative gene for a juvenile recessive form of amyotrophic lateral sclerosis (ALS2), 1 and a rare juvenile recessive form of primary lateral sclerosis (PLSJ) (1, 2). ALS2 is characterized by a loss of upper motor neurons and spasticity of limb and facial muscles occasionally associated with several signs of lower motor neuron defects (3), whereas PLSJ affects only upper motor neurons (4). Recently, several independent homozygous ALS2 mutations have been found in families segregating an infantile-onset ascending hereditary spastic paralysis (IAHSP) (5-7) and a single family of a recessive complicated hereditary spastic paraplegia (HSP) (8). Thus, ALS2 mutations account for a number of juvenile recessive motor neuron diseases, indicating that the ALS2 protein plays an important role in the maintenance and/or survival of motor neurons.The ALS2 gene encodes a protein of 1657 amino acid residues (aa), which contains three putative guanine nucleotide exchange factor (GEF) domains (1, 2). The N-terminal half of the ALS2 protein shares significant homology with RCC1 (regulator of chromosome condensation 1) (9), and this region is referred to as an RCC1-like domain (RLD), which has been found in a number of proteins (10 -13). Although RCC1 acts as a GEF for Ran (Ras-related nuclear) GTPase (9), the functions for RLD domains are still unclear. RLD is followed by a tandem organization of Dbl homology (...