Ligand-mediated endocytosis is an intricate regulatory mechanism for epidermal growth factor receptor (EGFR) signal transduction. Coordinated trafficking of EGFR ensures its temporal and spatial communication with downstream signaling effectors. We focused our work on Rab5, a monomeric GTPase shown to participate in early stages of the endocytic pathway. Rab5 has three isoforms (A, B, and C) sharing more than 90% of sequence identity. We individually ablated endogenous isoforms in HeLa cells with short interfering RNAs and examined the loss-of-function phenotypes. We found that suppression of Rab5A or 5B hampered the degradation of EGFR, whereas Rab5C depletion had very little effect. The differential delay of EGFR degradation also corresponds with retarded progression of EGFR from early to late endosomes. We investigated the activators/effectors of Rab5A that can potentially separate its potency in EGFR degradation from other isoforms and found that Rin1, a Rab5 exchange factor, preferably associated with Rab5A. Moreover, Rab5A activation is sensitive to EGF stimulation, and suppression of Rin1 diminished this sensitivity. Rabs are small molecular weight guanine nucleotide-binding proteins (G proteins) that specialize in regulating different stages of the intracellular membrane trafficking based on their subcellular localization and interacting protein scaffolds (2). The increasing number of Rab family members during evolution reflects ongoing specialization of membrane trafficking pathways. At least 63 Rabs have been identified in humans. Among these, Rab5 serves as the master regulator of the endocytic trafficking (3). It functions through recruitment of specific effector proteins involved in membrane tethering and docking (4 -8). The demonstration that at least 20 cytosolic proteins specifically interact with active Rab5 highlights the complexity of the downstream regulation by this GTPase and raises the possibility that Rab5 might also manage other aspects of the endosome function (4, 9). A common feature of the Rab family small GTPases is the existence of subgroups of structurally related isoforms sharing a high sequence identity (10). The Rab5 subgroup has three isoforms (A, B, and C) (3). A large scale mRNA expression profiling study from 79 human and 61 mouse nonredundant tissues shows distinct tissue distributions of the Rab5 isoforms, suggesting that the trafficking properties of the early endosomal network in developmentally distinct cell and tissue types is likely fine-tuned to fulfill subspecializations of a given pathway (10). In fact, several lines of evidence indicate that Rab5 isoforms can be functionally different. First, Rab5A is found to be the only isoform transcriptionally up-regulated in response to treatment with cytokines like interleukin-4 or interferon-␥ in macrophages (11, 12). Moreover, Rab5A but not Rab5C was found to be involved in the maturation of phagosomes containing Listeria monocytogenes (13). In primary hippocampal cultures, treatment with 3,5 dihydroxyphenylglycine, a grou...
Activated epidermal growth factor receptors (EGFRs) recruit intracellular proteins that mediate receptor signaling and endocytic trafficking. Rin1, a multifunctional protein, has been shown to regulate EGFR internalization (1). Here we show that EGF stimulation induces a specific, rapid, and transient membrane recruitment of Rin1 and that recruitment is dependent on the Src homology 2 (SH2) domain of Rin1. Immunoprecipitation of EGFR is accompanied by co-immunoprecipitation of Rin1 in a time-and ligand-dependent manner. Association of Rin1 and specifically the SH2 domain of Rin1 with the EGFR was dependent on tyrosine phosphorylation of the intracellular domain of the EGFR. The recruitment of Rin1, observed by light microscopy, indicated that although initially cytosolic, Rin1 was recruited to both plasma membrane and endosomes following EGF addition. Moreover, the expression of the SH2 domain of Rin1 substantially impaired the internalization of EGF without affecting internalization of transferrin. Finally, we found that Rin1 co-immunoprecipitated with a number of tyrosine kinase receptors but not with cargo endocytic receptors. These results indicate that Rin1 provides a link via its SH2 domain between activated tyrosine kinase receptors and the endocytic pathway through the recruitment and activation of Rab5a.
Objective We determined in patients with pulmonary arterial (PA) hypertension (PAH) whether in addition to increased production of elastase by PA smooth muscle cells (SMC) previously reported, PA elastic fibers are susceptible to degradation owing to their abnormal assembly. Approach and Results Fibrillin-1 and elastin are the major components of elastic fibers, and fibrillin-1 binds bone morphogenetic proteins (BMPs) and the large latent complex of transforming growth factor-β1 (TGFβ1). Thus, we considered whether BMPs like TGFβ1 contribute to elastic fiber assembly and whether this process is perturbed in PAH particularly when the BMP receptor, BMPR2, is mutant. We also assessed whether in mice with Bmpr2/1a compound heterozygosity, elastic fibers are susceptible to degradation. In PA SMC and adventitial fibroblasts (PAF), TGFβ1 increased elastin mRNA but the elevation in elastin protein, was dependent on BMPR2; TGFβ1 and BMP4, via BMPR2, increased extracellular accumulation of fibrillin-1. Both BMP4- and TGFβ1-stimulated elastic fiber assembly were impaired in idiopathic (I) PAH-PAF vs. control cells, particularly those with hereditary (H) PAH and a BMPR2 mutation. This was related to profound reductions in elastin and fibrillin-1 mRNA. Elastin protein was increased in IPAH PAF by TGFβ1 but only minimally so in BMPR2 mutant cells. Fibrillin-1 protein increased only modestly in IPAH or HPAH PAF stimulated with BMP4 or TGFβ1. In Bmpr2/1a heterozygote mice, reduced PA fibrillin-1 was associated with elastic fiber susceptibility to degradation and more severe pulmonary hypertension. Conclusion Disrupting BMPR2 impairs TGFβ1 and BMP4 mediated elastic fiber assembly and is of pathophysiologic significance in PAH.
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