Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin

Endophilin is a membrane-associated protein required for endocytosis of synaptic vesicles. Two models have been proposed for endophilin: that it alters lipid composition in order to shape membranes during endocytosis, or that it binds the polyphosphoinositide phosphatase synaptojanin and recruits this phosphatase to membranes. In this study, we demonstrate that the unc-57 gene encodes the Caenorhabditis elegans ortholog of endophilin A. We demonstrate that endophilin is required in C. elegans for synaptic vesicle recycling. Furthermore, the defects observed in endophilin mutants closely resemble those observed in synaptojanin mutants. The electrophysiological phenotype of endophilin and synaptojanin double mutants are virtually identical to the single mutants, demonstrating that endophilin and synaptojanin function in the same pathway. Finally, endophilin is required to stabilize expression of synaptojanin at the synapse. These data suggest that endophilin is an adaptor protein required to localize and stabilize synaptojanin at membranes during synaptic vesicle recycling.     

Regulation of clathrin-dependent endocytosis by diacylglycerol kinase delta: importance of kinase activity and binding to AP2alpha

DGKdelta (diacylglycerol kinase delta), which phosphorylates DAG (diacylglycerol) and converts it into PA (phosphatidic acid), has an important role in signal transduction. In the present study, we have demonstrated the molecular mechanism of DGKdelta-mediated regulation of clathrin-dependent endocytosis that controls the internalization, recycling and degradation of receptors. Involvement of DGKdelta in the regulation of clathrin-dependent endocytosis was previously proposed following genome-wide RNAi (RNA interference) screening. Clathrin-coated pits are mainly formed by clathrin and AP-2 (adaptor protein 2) complex. These proteins assemble a polyhedral lattice at the membrane and gather several endocytic accessory proteins. As the intracellular localization of DGKdelta2 overlapped with clathrin-coated pits, we predicted the possible regulation of clathrin-dependent endocytosis by DGKdelta2 and its interaction with some endocytosis-regulatory proteins. DGKdelta2 contained the DXF-type binding motifs, and DGKdelta2 bound to AP2alpha, a subunit of the AP-2 complex. DGKdelta2 interacted with the platform subdomain in the AP2alpha ear domain via F369DTFRIL and D746PF sequences in the catalytic domain of DGKdelta2. For further insight into the role for DGKdelta2 in clathrin-dependent endocytosis, we measured the transferrin and EGF (epidermal growth factor) uptake-expressing wild-type or mutant DGKdelta2 under knockdown of endogenous DGKdelta. Mutants lacking binding ability to AP2alpha as well as kinase-negative mutants could not compensate for the uptake of transferrin inhibited by siRNA (small interfering RNA) treatment, whereas overexpression of wild-type DGKdelta2 completely recovered the transferrin uptake. These results demonstrate that binding between DGKdelta2 and AP2alpha is involved in the transferrin internalization and that DGK activity is also necessary for the regulation of the endocytic process.     

Identification of Endophilins 1 and 3 as Selective Binding Partners for VGLUT1 and Their Co-Localization in Neocortical Glutamatergic Synapses: Implications for Vesicular Glutamate Transporter Trafficking and Excitatory Vesicle Formation

1. Selective protein–protein interactions between neurotransmitter transporters and their synaptic targets play important roles in regulating chemical neurotransmission. We screened a yeast two-hybrid library with bait containing the C-terminal amino acids of VGLUT1 and obtained clones that encode endophilin 1 and endophilin 3, proteins considered to play an integral role in glutamatergic vesicle formation.2. Using a modified yeast plasmid vector to enable more cost-effective screens, we analyzed the selectivity and specificity of this interaction. Endophilins 1 and 3 selectively recognize only VGLUT1 as the C-terminus of VGLUT2 and VGLUT3 do not interact with either endophilin isoform. We mutagenized four conserved stretches of primary sequence in VGLUT1 that includes two polyproline motifs (Pro1, PPAPPP, and Pro2, PPRPPPP), found only in VGLUT1, and two conserved stretches (SEEK, SYGAT), found also in VGLUT2 and VGLUT3. The absence of the VGLUT conserved regions does not affect VGLUT1–endophilin association. Of the two polyproline stretches, only one (Pro2) is required for binding specificity to both endophilin 1 and endophilin 3.3. We also show that endophilin 1 and endophilin 3 co-localize with VGLUT1 in synaptic terminals of differentiated rat neocortical neurons in primary culture. These results indicate that VGLUT1 and both endophilins are enriched in a class of excitatory synaptic terminals in cortical neurons and there, may interact to play an important role affecting the vesicular sequestration and synaptic release of glutamate.     

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.     

Protein Interactions of the Vesicular Glutamate Transporter VGLUT1

Exocytotic release of glutamate depends upon loading of the neurotransmitter into synaptic vesicles by vesicular glutamate transporters, VGLUTs. The major isoforms, VGLUT1 and 2, exhibit a complementary pattern of expression in synapses of the adult rodent brain that correlates with the probability of release and potential for plasticity. Indeed, expression of different VGLUT protein isoforms confers different properties of release probability. Expression of VGLUT1 or 2 protein also determines the kinetics of synaptic vesicle recycling. To identify molecular determinants that may be related to reported differences in VGLUT trafficking and glutamate release properties, we investigated some of the intrinsic differences between the two isoforms. VGLUT1 and 2 exhibit a high degree of sequence homology, but differ in their N- and C-termini. While the C-termini of VGLUT1 and 2 share a dileucine-like trafficking motif and a proline-, glutamate-, serine-, and threonine-rich PEST domain, only VGLUT1 contains two polyproline domains and a phosphorylation consensus sequence in a region of acidic amino acids. The interaction of a VGLUT1 polyproline domain with the endocytic protein endophilin recruits VGLUT1 to a fast recycling pathway. To identify trans-acting cellular proteins that interact with the distinct motifs found in the C-terminus of VGLUT1, we performed a series of in vitro biochemical screening assays using the region encompassing the polyproline motifs, phosphorylation consensus sites, and PEST domain. We identify interactors that belong to several classes of proteins that modulate cellular function, including actin cytoskeletal adaptors, ubiquitin ligases, and tyrosine kinases. The nature of these interactions suggests novel avenues to investigate the modulation of synaptic vesicle protein recycling.     

The Clathrin Adaptor Proteins ARH,Dab2, and Numb Play Distinct Roles in Niemann-Pick C1-Like 1 Versus Low Density Lipoprotein Receptor-mediated Cholesterol Uptake

The uptake of circulating low density lipoproteins (LDL) is mediated by LDL receptor (LDLR) through clathrin-dependent endocytosis. At the early stage of this process, adaptor proteins ARH and Dab2 specifically bind the endocytic signal motif in LDLR and recruit clathrin/AP2 to initiate internalization. On the other hand, intestinal cholesterol is absorbed by Niemann-Pick C1-Like 1 (NPC1L1) through clathrin-dependent endocytosis. Another adaptor protein, Numb recognizes the endocytic motif in NPC1L1 C terminus and couples NPC1L1 to endocytic machinery. The ARH, Dab2, and Numb proteins contain a homogeneous phosphotyrosine binding (PTB) domain that directly binds endocytic motifs. Because ARH, Dab2, and Numb are all PTB domain family members, the emerging mystery is whether these adaptors act complementally in LDLR and NPC1L1 endocytosis. Here, we found that ARH and Dab2 did not bind NPC1L1 and were not required for NPC1L1 internalization. Similarly, Numb lacked the ability to interact with the LDLR C terminus and was dispensable for LDL uptake. Only the Numb isoforms with shorter PTB domain could facilitate NPC1L1 endocytosis. Besides the reported function in intestinal cholesterol absorption, Numb also mediated cholesterol reabsorption from bile in liver. We further identified a Numb variant with G595D substitution in humans of low blood LDL-cholesterol. The G595D substitution impaired NPC1L1 internalization and cholesterol reabsorption, due to attenuating affinity of Numb to clathrin/AP2. These results demonstrate that Numb specifically regulates NPC1L1-mediated cholesterol absorption both in human intestine and liver, distinct from ARH and Dab2, which selectively participate in LDLR-mediated LDL uptake.     

Ataxin-2 associates with the endocytosis complex and affects EGF receptor trafficking

Ataxin-2 is a novel protein, where the unstable expansion of an internal polyglutamine domain can cause the neurodegenerative disease Spinocerebellar Ataxia type 2 (SCA2). To elucidate its cellular function, we have used full-length ataxin-2 as bait in a yeast two-hybrid screen of human adult brain cDNA. As binding partners we found endophilin A1 and A3, two brain-expressed members of the endophilin A family involved in synaptic vesicle endocytosis. Co-immunoprecipitation studies confirmed the binding of these proteins as an endogenous complex in mouse brain. In vitro binding experiments narrowed the binding interfaces down to two proline-rich domains on ataxin-2, which interacted with the SH3 domain of endophilin A1/A3. Ataxin-2 and endophilin associated at the endoplasmic reticulum as well as at the plasma membrane as determined by immunofluorescence microscopy of transfected cell lines, and by centrifugation fractionation studies of mouse brain. Importantly, the pattern observed in transfected cells was conserved in rat hippocampal neurons. In the mouse brain, an association of ataxin-2 with endocytic proteins such as the adaptor CIN85 and the ubiquitin ligase c-Cbl was also demonstrated. GST pull-down assays showed ataxin-2 to directly interact with the SH3 domains A and C of CIN85 and with the SH3 domain of Src, a kinase activated after receptor stimulation. Functional studies demonstrated that ataxin-2 affects endocytic trafficking of the epidermal growth factor receptor (EGFR). Taken together, these data implicate ataxin-2 to play a role in endocytic receptor cycling.     

What is the function of neuronal AP‐3?

Neurotransmission requires the proper organization and rapid recycling of synaptic vesicles. Rapid retrieval has been suggested to occur either by kiss-and-stay or kiss-and-run mechanisms, whereas classical recycling is mediated by clathrin-dependent endocytosis. Molecular coats are key components in the selection of cargos, AP-2 (adaptor protein 2) playing a prominent role in synaptic vesicle endocytosis. Another coat protein, AP-3, has been implicated in synaptic vesicle biogenesis and in the generation of secretory and lysosomal-related organelles. In the present review, we will particularly focus on the recent data concerning the recycling of synaptic vesicles and the function of AP-3 and the v-SNARE (vesicular soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) TI-VAMP (tetanus neurotoxin-insensitive vesicle-associated membrane protein) in these processes. We propose that AP-3 plays an important regulatory role in neurons which contributes to the basal and stimulated exocytosis of synaptic vesicles.     

Signalling through phospholipase C interferes with clathrin-mediated endocytosis

We investigated if phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) hydrolysis by phospholipase C activation through cell surface receptors would interfere with clathrin-mediated endocytosis as recruitment of clathrin assembly proteins is PtdIns(4,5)P2-dependent. In the WKPT renal epithelial cell line, endocytosed insulin and beta2-glycoprotein I (beta2gpI) were observed in separate compartments, although endocytosis of both ligands was clathrin-dependent as demonstrated by expression of the clathrin-binding C-terminal domain of AP180 (AP180-C). The two uptake mechanisms were different as only insulin uptake was reduced when the mu2-subunit of the adaptor complex AP-2 was silenced by RNA interference. ATP receptors are expressed at the apical surface of renal cells and, thus, we examined the effect of extracellular ATP on insulin and beta2gpI uptake. ATP stimulated phospholipase C activity, and also suppressed uptake of insulin, but not beta2gpI. This effect was reversed by the PLC inhibitor U-73122. In polarized cell cultures, insulin uptake was apical, whereas beta2gpI uptake was through the basolateral membrane, thus providing an explanation for selective inhibition of insulin endocytosis by ATP. Taken together, these results demonstrate that stimulation of apical G-protein-coupled P2Y receptors, which are coupled to phospholipase C activation diminishes clathrin-mediated endocytosis without interfering with basolateral endocytic mechanisms.     

Polarized trafficking and surface expression of the AQP4 water channel are coordinated by serial and regulated interactions with different clathrin-adaptor complexes.

Aquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to specific membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also specifically expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane trafficking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosine-based basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the mu subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in mu subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4-mu 3A interaction and enhancing AQP4-lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression.     

吞蛋白A2在非网格蛋白内吞中的作用研究进展

内吞作用是细胞从细胞外空间和内化横跨膜的细胞表面蛋白转运物质到细胞内的过程.吞蛋白(endophilin)一直被认为参与了网格蛋白介导的细胞内吞作用,2015年《自然》(Nature)发表的两篇研究论文报道了一种由endophilin A标记和控制的独立于网格蛋白的有被囊泡内吞作用.本文主要综述近年来endophilin A2的研究,着重介绍endophilin A2在非网格蛋白介导的内吞作用中的功能和机制.     

Stonin 2 is an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling

Clathrin-mediated endocytosis is involved in the internalization, recycling, and degradation of cycling membrane receptors as well as in the biogenesis of synaptic vesicle proteins. While many constitutively internalized cargo proteins are recognized directly by the clathrin adaptor complex AP-2, stimulation-dependent endocytosis of membrane proteins is often facilitated by specialized sorting adaptors. Although clathrin-mediated endocytosis appears to be a major pathway for presynaptic vesicle cycling, no sorting adaptor dedicated to synaptic vesicle membrane protein endocytosis has been indentified in mammals. Here, we show that stonin 2, a mammalian ortholog of Drosophila stoned B, facilitates clathrin/AP-2-dependent internalization of synaptotagmin and targets it to a recycling vesicle pool in living neurons. The ability of stonin 2 to facilitate endocytosis of synaptotagmin is dependent on its association with AP-2, an intact mu-homology domain, and functional AP-2 heterotetramers. Our data identify stonin 2 as an AP-2-dependent endocytic sorting adaptor for synaptotagmin internalization and recycling.     

Endocytic Structures and Synaptic Vesicle Recycling at a Central Synapse in Awake Rats

The synaptic vesicle (SV) cycle has been studied extensively in cultured cells and slice preparations, but not much is known about the roles and relative contributions of endocytic pathways and mechanisms of SV recycling in vivo, under physiological patterns of activity. We employed horseradish peroxidase (HRP) as an in vivo marker of endocytosis at the calyx of Held synapse in the awake rat. Ex vivo serial section scanning electron microscopy and 3D reconstructions revealed two categories of labelled structures: HRP‐filled SVs and large cisternal endosomes. Inhibition of adaptor protein complexes 1 and 3 (AP‐1, AP‐3) by in vivo application of Brefeldin A (BFA) disrupted endosomal SV budding while SV recycling via clathrin‐mediated endocytosis (CME) remained unaffected. In conclusion, our study establishes cisternal endosomes as an intermediate of the SV cycle and reveals CME and endosomal budding as the predominant mechanisms of SV recycling in a tonically active central synapse in vivo.     

Interplay between VGLUT isoforms and endophilin A1 regulates neurotransmitter release and short-term plasticity

Vesicular glutamate transporters (VGLUTs) are essential for filling synaptic vesicles with glutamate and mammals express three VGLUT isoforms (VGLUT1-3) with distinct spatiotemporal expression patterns. Here, we find that neurons expressing VGLUT1 have lower release probability and less short-term depression than neurons expressing VGLUT2 or VGLUT3. Investigation of the underlying mechanism identified endophilin A1 as a positive regulator of exocytosis whose expression levels are positively correlated with release efficiency and showed that the differences in release efficiency between VGLUT1- and VGLUT2-expressing neurons are due to VGLUT1's ability to bind endophilin A1 and inhibit endophilin-induced enhancement of release probability.     

A Tandem Di-hydrophobic Motif Mediates Clathrin-dependent Endocytosis via Direct Binding to the AP-2 ασ2 Subunits

Select plasma membrane proteins can be marked as cargo for inclusion into clathrin-coated pits by common internalization signals (e.g. YXXΦ, dileucine motifs, NPXY) that serve as universal recognition sites for the AP-2 adaptor complex or other clathrin-associated sorting proteins. However, some surface proteins, such as the Kir2.3 potassium channel, lack canonical signals but are still targeted for clathrin-dependent endocytosis. Here, we explore the mechanism. We found an unusual endocytic signal in Kir2.3 that is based on two consecutive pairs of hydrophobic residues. Characterized by the sequence ΦΦXΦΦ (a tandem di-hydrophobic (TDH) motif, where Φ is a hydrophobic amino acid), the signal shows no resemblance to other endocytic motifs, yet it directly interacts with AP-2 to target the Kir2.3 potassium channel into the endocytic pathway. We found that the tandem di-hydrophobic motif directly binds to the ασ2 subunits of AP-2, interacting within a large hydrophobic cleft that encompasses part of the docking site for di-Leu signals, but includes additional structures. These observations expand the repertoire of clathrin-dependent internalization signals and the ways in which AP-2 can coordinate endocytosis of cargo proteins.     

Endocytosis of Ubiquitylation‐Deficient EGFR Mutants via Clathrin‐Coated Pits is Mediated by Ubiquitylation

Signaling by epidermal growth factor receptor (EGFR) is controlled by endocytosis. However, mechanisms of EGFR endocytosis remain poorly understood. Here, we found that the EGFR mutant lacking known ubiquitylation, acetylation and clathrin adaptor AP‐2‐binding sites (21KRΔAP2) was internalized at relatively high rates via the clathrin‐dependent pathway in human duodenal adenocarcinoma HuTu‐80 cells. RNA interference analysis revealed that this residual internalization is strongly inhibited by depletion of Grb2 and the E2 ubiquitin‐conjugating enzyme UbcH5b/c, and partially affected by depletion of the E3 ubiquitin ligase Cbl and ubiquitin‐binding adaptors, indicating that an ubiquitylation process is involved. Several new ubiquitin conjugation sites were identified by mass spectrometry in the 21KRΔAP2 mutant, suggesting that cryptic ubiquitylation may mediate endocytosis of this mutant. Total internal reflection fluorescence microscopy imaging of HuTu‐80 cells transfected with labeled ubiquitin adaptor epsin1 demonstrated that the ubiquitylation‐deficient EGFR mutant was endocytosed through a limited population of epsin‐enriched clathrin‐coated pits (CCPs), although with a prolonged CCP lifetime. Native EGFR was recruited with the same efficiency into CCPs containing either AP‐2 or epsin1 that were tagged with fluorescent proteins by genome editing of MDA‐MD‐231 cells. We propose that two redundant mechanisms, ubiquitylation and interaction with AP‐2, contribute to EGFR endocytosis via CCPs in a stochastic fashion.      

Decoupling of activation and effector binding underlies ARF6 priming of fast endocytic recycling

The small GTP-binding protein ADP-ribosylation factor 6 (ARF6) controls the endocytic recycling pathway of several plasma membrane receptors. We analyzed the localization and GDP/GTP cycle of GFP-tagged ARF6 by total internal reflection fluorescent microscopy. We found that ARF6-GFP associates with clathrin-coated pits (CCPs) at the plasma membrane in a GTP-dependent manner in a mechanism requiring the adaptor protein complex AP-2. In CCP, GTP-ARF6 mediates the recruitment of the ARF-binding domain of downstream effectors including JNK-interacting proteins 3 and 4 (JIP3 and JIP4) after the burst recruitment of the clathrin uncoating component auxilin. ARF6 does not contribute to receptor-mediated clathrin-dependent endocytosis. In contrast, we found that interaction of ARF6 and JIPs on endocytic vesicles is required for trafficking of the transferrin receptor in the fast, microtubule-dependent endocytic recycling pathway. Our findings unravel a novel mechanism of separation of ARF6 activation and effector function, ensuring that fast recycling may be determined at the level of receptor incorporation into CCPs.     

SMAP2, a novel ARF GTPase-activating protein, interacts with clathrin and clathrin assembly protein and functions on the AP-1-positive early endosome/trans-Golgi network

We recently reported that SMAP1, a GTPase-activating protein (GAP) for Arf6, directly interacts with clathrin and regulates the clathrin-dependent endocytosis of transferrin receptors from the plasma membrane. Here, we identified a SMAP1 homologue that we named SMAP2. Like SMAP1, SMAP2 exhibits GAP activity and interacts with clathrin heavy chain (CHC). Furthermore, we show that SMAP2 interacts with the clathrin assembly protein CALM. Unlike SMAP1, however, SMAP2 appears to be a regulator of Arf1 in vivo, because cells transfected with a GAP-negative SMAP2 mutant were resistant to brefeldin A. SMAP2 colocalized with the adaptor proteins for clathrin AP-1 and EpsinR on the early endosomes/trans-Golgi-network (TGN). Moreover, overexpression of SMAP2 delayed the accumulation of TGN38/46 molecule on the TGN. This suggests that SMAP2 functions in the retrograde, early endosome-to-TGN pathway in a clathrin- and AP-1-dependent manner. Thus, the SMAP gene family constitutes an important ArfGAP subfamily, with each SMAP member exerting both common and distinct functions in vesicle trafficking.     

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses

Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.     

EHDS are serine phosphoproteins: EHD1 phosphorylation is enhanced by serum stimulation

Endocytic processes are mediated by multiple protein-protein interacting modules and regulated by phosphorylation and dephosphorylation. The Eps15 homology domain containing protein 1 (EHD1) has been implicated in regulating recycling of proteins, internalized both in clathrin-dependent and clathrin-independent endocytic pathways, from the recycling compartment to the plasma membrane. EHD1 was found in a complex with clathrin, adaptor protein complex-2 (AP-2) and insulin-like growth factor-1 receptor (IGF-1R), and was shown to interact with Rabenosyn-5, SNAP29, EHBP1 (EH domain binding protein 1) and syndapin I and II. In this study, we show that EHD1, like the other human EHDs, undergoes serine-phosphorylation. Our results also indicate that EHD1 is a serum-inducible serine-phosphoprotein and that PKC (protein kinase C) is one of its kinases. In addition, we show that inhibitors of clathrin-mediated endocytosis decrease EHD1 phosphorylation, while inhibitors of caveolinmediated endocytosis do not affect EHD1 phosphorylation. The results of experiments in which inhibitors of endocytosis were employed strongly suggest that EHD1 phosphorylation occurs between early endosomes and the endocytic recycling compartment.