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  • Ligand binding RTK activation induces the


    Ligand binding (RTK activation) induces the endocytic internalization of RTKs, and this has been demonstrated for CSF-1R [15], [16], [17]. Endocytosed RTKs are targeted to the lysosome, which we and others have established to be dependent on ubiquitination via CBL-family and recognition by ESCRT complexes [15], [18], [19], [20], [21]. Internalization and degradation of RTKs have classically been thought to be a mechanism for cellular attenuation of RTK signalling. However, activated RTKs are known to signal in endosomes and may require endocytosis to transduce specific signals [22]. Recently, Erk1/2 activation due to CSF-1R signalling has been shown to require endocytosis, while signalling at the cell surface was sufficient for STAT pathway activation [23]. Compared to intracellular trafficking of ligand-stimulated RTKs, the trafficking of unstimulated/basal RTKs is less understood. In the absence of CSF-1, CSF-1R has been shown to undergo constitutive internalization and basal recycling at a relatively low rate [24]. An understanding of the mechanisms of RTK traffic in the absence of ligand-induced activation is of fundamental importance as these mechanisms, together with the anterograde transport of newly synthesized protein, help determine the level of the activation-ready, cell surface pool of RTKs. Such mechanisms can also determine the kinetics of restoration of cell surface RTK display following ligand-induced degradation for subsequent ligand binding and activation. Yet, little is known about these basic mechanisms of RTK traffic, and CSF-1R trafficking in macrophages has not been reported. The four members of the Eps15-homology domain-containing (EHD) protein family have emerged as regulators of cellular receptor trafficking [25]. Structurally, EHD proteins are characterized by highly related primary amino Liraglutide sequences and conserved domain structure amongst family members: a nucleotide-binding G-domain that folds similarly to the GTPase dynamin, but instead hydrolyzes ATP; coiled-coiled regions that form a membrane lipid-binding domain; and an EH domain near the C-terminus that mediates interactions with partner proteins by binding to asparagine-proline-phenylalanine (NPF)-containing motifs [26]. In vitro studies have established that EHD proteins function similarly to dynamin as scission proteins to promote vesicular budding involved in cellular trafficking of receptors [27]. While biochemical roles of EHD proteins have been elucidated in some detail, their physiological roles are just now being unraveled. Our laboratory has previously utilized mouse models to understand the physiological functions of EHD family proteins [28], [29], [30], [31], [32], [33], [34]. We have found that EHD1 deletion on Liraglutide a mixed C57BL/6 and 129Sv background is partially penetrant embryonic lethal and associated with defective spermatogenesis and lens developmental defects, while it is fully embryonic lethal on a predominantly C57BL/6 background [35], [36], [37]. Here, we use primary bone marrow-derived macrophages (BMDMs) with inducible EHD1-Knockout (EHD1-KO) capacity, to study the biological and physiological function of EHD1 in macrophages. Here, we report EHD1 is required for delivery and display of CSF-1R upon the cell surface, ensuring CSF-1R activation/signalling, and downstream macrophage functional response.
    Materials & methods
    Discussion CSF-1R is essential for the development and varied functions of monocyte-macrophage lineage cells [2]. Abnormal CSF-1R-dependent macrophage functions contribute to variety of human diseases and aberrant CSF-1R signalling is a contributor to oncogenesis [3], [11], [48], [49]. A pre-requisite for all CSF-1R-mediated macrophage biological functions is the display of the receptor at the cell surface in order for ligand to bind and initiate signal transduction cascades. Mechanisms that govern the post-synthesis/maturation traffic of CSF-1R to the cell surface are therefore biologically pivotal, yet nothing is known about these mechanisms at the present time. Here, we used primary bone marrow-derived macrophages (BMDMs) derived from an inducible knockout mouse model to provide evidence that EHD1, a member of the EHD family of endocytic recycling regulators, serves a novel and critical role in ensuring Golgi to cell surface traffic of CSF-1R. We show that this novel function of EHD1 in delivering CSF-1R from the Golgi to the membrane. In the absence of EHD1, CSF-1R is shunted to the lysosome. Our studies show that EHD1-dependent cell surface transport of CSF-1R ensures subsequent ligand-induced cellular activation. Our findings that EHD1 functions as a novel regulator CSF-1R transport to the cell surface help assign a new functional role to EHD1.