RESEARCH ARTICLE NECAP2 controls clathrin coat recruitment to early endosomes for fast endocytic recycling John P. Chamberland, Lauren T. Antonow, Michel Dias Santos and Brigitte Ritter* ABSTRACT Endocytic recycling returns receptors to the plasma membrane following internalization and is essential to maintain receptor levels on the cell surface, re-sensitize cells to extracellular ligands and for continued nutrient uptake. Yet, the protein machineries and mechanisms that drive endocytic recycling remain ill-defined. Here, we establish that NECAP2 regulates the endocytic recycling of EGFR and transferrin receptor. Our analysis of the recycling dynamics revealed that NECAP2 functions in the fast recycling pathway that directly returns cargo from early endosomes to the cell surface. In contrast, NECAP2 does not regulate the clathrin-mediated endocytosis of these cargos, the degradation of EGFR or the recycling of transferrin along the slow, Rab11-dependent recycling pathway. We show that protein knockdown of NECAP2 leads to enlarged early endosomes and causes the loss of the clathrin adapter AP-1 from the organelle. Through structure-function analysis, we define the protein-binding interfaces in NECAP2 that are crucial for AP-1 recruitment to early endosomes. Together, our data identify NECAP2 as a pathway-specific regulator of clathrin coat formation on early endosomes for fast endocytic recycling. KEY WORDS: EGFR, Rab4, Clathrin, Early endosomes, Endosomal sorting, Fast recycling INTRODUCTION Receptor levels at the cell surface are dynamically regulated to control numerous cellular functions, including receptor signaling, nutrient uptake and cell migration. To remove membrane-bound molecules and receptor–ligand complexes from the surface, these cargos are internalized through clathrin-mediated and clathrin- independent endocytosis. Internalized receptors then enter early endosomes, which function as the key sorting station in deciding the fate of receptor cargo. Ubiquitylated receptors engage the ESCRT complex for sorting into multivesicular bodies and subsequent receptor degradation (Raiborg and Stenmark, 2009). The degradative pathway is crucial for the termination of signaling cascades downstream of ligand-activated receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR). In contrast, endocytic recycling returns receptors to the cell surface, thereby replenishing the surface pool of receptors to re-sensitize cells to extracellular ligands, to maintain nutrient supply and to promote cell motility (Grant and Donaldson, 2009; Kelly and Owen, 2011; Maritzen et al., 2015). Early endosomal sorting and endocytic recycling are regulated by members of the Arf and Rab families of small GTPases. Activation of Rabs and Arfs by guanine-nucleotide exchange factors (GEFs) enables the GTP-bound GTPases to recruit downstream effector proteins that fulfill pathway-specific functions and promote the formation of cargo-carrying vesicles and tubules. For example, Rab35 mediates cadherin recycling, whereas Arf6 controls β1-integrin recycling (Allaire et al., 2013). Transferrin receptor (TfnR) is the canonical marker of endocytic recycling, and transferrin remains bound to the receptor until TfnR reaches the cell surface. Notably, TfnR enters into recycling pathways with differential kinetics (Sheff et al., 1999). The fast recycling pathway directly returns TfnR from early endosomes to the cell surface and is regulated by Rab4a (van der Sluijs et al., 1992). In the slow recycling pathway, Rab4b-mediated sorting sends TfnR from early to Rab11a-positive recycling endosomes for subsequent transport to the cell surface (Bhuin and Roy, 2015; Perrin et al., 2013; Welz et al., 2014). The fast and slow recycling pathways both recruit the clathrin adapter complex AP-1 and clathrin, albeit through different mechanisms. Rab4b directly recruits AP-1 as an effector to promote cargo sorting from early to recycling endosomes (Perrin et al., 2013). Rab4a instead triggers a multistep GTPase cascade that, in addition to AP-1, also recruits the clathrin adapters AP-3 and GGA3 to early endosomes (D’Souza et al., 2014). It is currently unclear whether the variety of clathrin adapters recruited by Rab4a represents a functional diversity in Rab4a-mediated recycling. However, the formation of recycling vesicles and tubules are likely to require a larger protein machinery beyond the clathrin adapters and clathrin themselves, similar to the complexity seen for clathrin- mediated endocytosis (Kirchhausen et al., 2014; McMahon and Boucrot, 2011). The characterization of the protein machineries driving endocytic recycling will be crucial for the understanding of the functional divergence between different recycling pathways and their role in normal cell function. We have previously identified the adaptin ear-binding coat- associated protein (NECAP) family during our proteomics analysis of clathrin-coated vesicles from adult rat brain (Blondeau et al., 2004; Ritter et al., 2003). The two members of the family, NECAP1 and NECAP2, share a high degree of sequence and structural similarity. At their N-terminus, the NECAPs encode a pleckstrin homology (PH)-like domain that functions as a protein-binding module (Ritter et al., 2007). In addition, NECAPs display peptide motifs at their C-terminus that promote interactions with the clathrin adaptor complexes AP-1 and AP-2 (Ritter et al., 2004). Our functional studies of NECAP1 have revealed that NECAP1 functions together with AP-2 in clathrin-mediated endocytosis at the cell surface (Ritter et al., 2013). In fact, the NECAP1 PH domain binds to some of the same accessory proteins that interact with AP-2 such that NECAP1 and AP-2 cooperate in the recruitment of accessory proteins to the site of vesicle formation (Ritter et al., Received 28 April 2015; Accepted 19 May 2016 Boston University School of Medicine, Biochemistry Department, Boston, MA 02118, USA. *Author for correspondence (britter@bu.edu) J.P.C., 0000-0002-7862-1371; L.T.A., 0000-0002-9622-4994; M.D.S., 0000- 0002-5976-7973; B.R., 0000-0002-0094-0460 2625 © 2016. Published by The Company of Biologists Ltd | Journal of Cell Science (2016) 129, 2625-2637 doi:10.1242/jcs.173708 Journal of Cell Science