Necl-1/CADM3 regulates cone synapse formation in the mouse retina.

In vertebrates, retinal neural circuitry for visual perception is organized in specific layers. The outer plexiform layer is the first synaptic region in the visual pathway, where photoreceptor synaptic terminals connect with bipolar and horizontal cell processes. However, molecular mechanisms underlying cone synapse formation to mediate OFF pathways remain unknown. This study reveals that Necl-1/CADM3 is localized at S- and S/M-opsin-containing cones and dendrites of type 4 OFF cone bipolar cells (CBCs). In Necl-1-/- mouse retina, synapses between cones and type 4 OFF CBCs were dislocated, horizontal cell distribution became abnormal, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors were dislocated. Necl-1-/- mice exhibited aberrant short-wavelength-light-elicited signal transmission from cones to OFF CBCs, which was rescued by AMPA receptor potentiator. Additionally, Necl-1-/- mice showed impaired optokinetic responses. These findings suggest that Necl-1 regulates cone synapse formation to mediate OFF cone pathways elicited by short-wavelength light in mouse retina.

Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain.

Astrocytes interact with not only synapses but also brain blood vessels through perivascular astrocyte endfeet (PV-AEF) to form the neurovascular unit (NVU). However, PV-AEF components have not been fully identified. Here, we biochemically isolated blood vessels from mouse brain homogenates and purified PV-AEF. The purified PV-AEF were observed in different sizes, similar to PV-AEF on brain blood vessels. Mass spectrometry analysis identified 9,762 proteins in the purified PV-AEF, including cell adhesion molecules, nectin-2δ, Kirrel2, and podoplanin. Immunofluorescence microscopic analysis revealed that nectin-2δ and podoplanin were concentrated mainly in arteries/arterioles and veins/venules of the mouse brain, whereas Kirrel2 was mainly in arteries/arterioles. Nectin-2α/δ, Kirrel2, and podoplanin were preferentially observed in large sizes of the purified PV-AEF. Furthermore, Kirrel2 potentially has cell adhesion activity of cultured astrocytes. Collectively, these results indicate that PV-AEF have heterogeneity in sizes and molecular components, implying different roles of PV-AEF in NVU function depending on vascular regions.

s-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons.

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.

Necl2/3-mediated mechanism for tripartite synapse formation.

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

Identification of lysophosphatidic acid in serum as a factor that promotes epithelial apical junctional complex organization.

The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization-promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol-novel PKC and Rho-ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho-ROCK pathway activation-mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization-disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization-promoting activity in a manner dependent on or independent of AJ proteins.

Stimulatory role of nectin-4 and p95-ErbB2 in multilayered T47D cell proliferation.

Multilayered proliferation in an adherent culture as well as proliferation in a suspension culture is a characteristic feature of cancer cells. We previously showed using T47D human mammary cancer cells that nectin-4, upregulated in many cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2ΔEx16, and enhances DNA synthesis mainly through the PI3K-AKT pathway in an adherent culture. We showed here that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and ErbB2 or that of nectin-4 and ErbB2ΔEx16, cooperatively enhanced multilayered T47D cell proliferation through the Hippo pathway-mediated SOX2 gene expression in an adherent culture. T47D cells expressed the components of the apical junctional complex (AJC) consisting of adherens junctions (AJs) and tight junctions and cell polarity molecules, but not the AJ component afadin. The AJC and apicobasal polarity were disorganized in T47D cells in a monolayer and T47D cells stably expressing both nectin-4 and p95-ErbB2 in multilayers. These results indicate that nectin-4 and p95-ErbB2 play a stimulatory role in multilayered proliferation in an adherent culture.

Nectin-2 in general and in the brain.

Nectins are immunoglobulin-like cell adhesion molecules constituting a family with four members, nectin-1, nectin-2, nectin-3, and nectin-4. In the brain, nectin-2 as well as nectin-1 and nectin-3 are expressed whereas nectin-4 is hardly expressed. In the nervous system, physiological functions of nectin-1 and nectin-3, such as synapse formation, mossy fiber trajectory regulation, interneurite affinity, contextual fear memory formation, and stress-related mental disorders, have been revealed. Nectin-2 is ubiquitously expressed in non-neuronal tissues and various nectin-2 functions in non-nervous systems have been extensively investigated, but nectin-2 functions in the brain have not been revealed until recently. Recent findings have revealed that nectin-2 is expressed in the specific areas of the brain and plays important roles, such as homeostasis of astrocytes and neurons and the formation of synapses. Moreover, a single nucleotide polymorphism in the human NECTIN2 gene is associated with Alzheimer's disease. We here summarize recent progress in our understanding of nectin-2 functions in the brain.

A novel role for PRL in regulating epithelial cell density by inducing apoptosis at confluence.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-ß pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.

Nectins and Nectin-like molecules in synapse formation and involvement in neurological diseases.

Synapses are interneuronal junctions which form neuronal networks and play roles in a variety of functions, including learning and memory. Two types of junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAJs), have been identified. SJs are found at all excitatory and inhibitory synapses whereas PAJs are found at excitatory synapses, but not inhibitory synapses, and particularly well developed at hippocampal mossy fiber giant excitatory synapses. Both SJs and PAJs are mediated by cell adhesion molecules (CAMs). Major CAMs at SJs are neuroligins-neurexins and Nectin-like molecules (Necls)/CADMs/SynCAMs whereas those at PAJs are nectins and cadherins. In addition to synaptic PAJs, extrasynaptic PAJs have been identified at contact sites between neighboring dendrites near synapses and regulate synapse formation. In addition to SJs and PAJs, a new type of cell adhesion apparatus different from these junctional apparatuses has been identified and named nectin/Necl spots. One nectin spot at contact sites between neighboring dendrites at extrasynaptic regions near synapses regulates synapse formation. Several members of nectins and Necls had been identified as viral receptors before finding their physiological functions as CAMs and evidence is accumulating that many nectins and Necls are related to onset and progression of neurological diseases. We review here nectin and Necls in synapse formation and involvement in neurological diseases.

Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula.

The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and ß-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, ß-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.

Afadin regulates actomyosin organization through αE-catenin at adherens junctions.

Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with ß-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with ß-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with ß-catenin. Although a "catch-bond model" partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with ß-catenin and enhances its F-actin-binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with ß-catenin and E-cadherin at adherens junctions.

Identification of Sox2 and NeuN Double-Positive Cells in the Mouse Hypothalamic Arcuate Nucleus and Their Reduction in Number With Aging.

The hypothalamus plays a central role in homeostasis and aging. The hypothalamic arcuate nucleus (ARC) controls homeostasis of food intake and energy expenditure and retains adult neural stem cells (NSCs)/progenitor cells. Aging induces the loss of NSCs and the enhancement of inflammation, including the activation of glial cells in the ARC, but aging-associated alterations of the hypothalamic cells remain obscure. Here, we identified Sox2 and NeuN double-positive cells in a subpopulation of cells in the mouse ARC. These cells were reduced in number with aging, although NeuN-positive neuronal cells were unaltered in the total number. Diet-induced obesity mice fed with high-fat diet presented a similar hypothalamic alteration to aged mice. This study provides a new insight into aging-induced changes in the hypothalamus.

Nectin-4 cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, enhancing their activation and DNA synthesis.

Nectin-4 cell adhesion molecule and ErbB2 tyrosine kinase receptor are upregulated in many cancers, including breast cancer, and promote cancer cell proliferation and metastasis. Using human breast cancer cell lines T47D and SUM190-PT, in which both nectin-4 and ErbB2 were upregulated, we showed here that nectin-4 cis-interacted with ErB2 and enhanced its dimerization and activation, followed by the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. The third immunoglobulin-like domain of nectin-4 cis-interacted with domain IV of ErbB2. This region differs from the trastuzumab-interacting region but is included in the trastuzumab-resistant splice variants of ErbB2, p95-ErbB2 and ErbB2ΔEx16. Nectin-4 also cis-interacted with these trastuzumab-resistant splice variants and enhanced the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. In addition, nectin-4 enhanced the activation of the p95-ErbB2-induced JAK-STAT3 signalling pathway, but not the ErbB2- or ErbB2ΔEx16-induced JAK-STAT3 signalling pathway. These results indicate that nectin-4 cis-interacts with ErbB2 and its trastuzumab-resistant splice variants and enhances the activation of these receptors and downstream signalling pathways in a novel mechanism.

Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula.

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.

Requirement of the F-actin-binding activity of l-afadin for enhancing the formation of adherens and tight junctions.

The apical junctional complex consists of adherens junctions (AJs) and tight junctions (TJs) in polarized epithelial cells, which are attached to each other to form a sheet. Actin filaments (F-actin) are associated with AJs and TJs and required for the formation and maintenance of this complex. l-Afadin is an F-actin-binding protein, which is localized at AJs through binding to the cell adhesion molecule nectin, and regulates the formation of AJs and TJs. However, the role of the F-actin-binding activity of l-afadin for the formation of the apical junctional complex remains unknown. We generated here the cultured EpH4 mouse mammary epithelial cells in which afadin was genetically ablated. In the Ca2+ switch assay, the formation of both AJs and TJs was markedly impaired in the afadin-deficient cells. Re-expression of l-afadin in the afadin-deficient cells fully restored the formation of both AJs and TJs, but the re-expression of the l-afadin mutant lacking the FAB domain did not completely restore the formation of AJs or TJs. These results indicate that the F-actin-binding activity of l-afadin is required for enhancing the formation of both AJs and TJs.

Roles of the third Ig-like domain of Necl-5/PVR and the fifth Ig-like domain of the PDGF receptor in its signaling.

The immunoglobulin (Ig)-like cell adhesion molecule nectin-like molecule (Necl)-5/poliovirus receptor is up-regulated in many types of cancer cells and implicated in their abnormally enhanced cell proliferation and movement. We previously showed that Necl-5 cis-interacts with the platelet-derived growth factor (PDGF) receptor ß through the extracellular region and enhances its signaling. Although this cis-interaction does not affect the PDGF-induced tyrosine phosphorylation of the receptor, the interaction of the cytoplasmic region of Necl-5 with sprouty2 and the regulation of its activity are required for the enhancement of the PDGF receptor ß signaling by Necl-5. We investigated here the more detailed mechanism for this cis-interaction of Necl-5 with the PDGF receptor ß. Necl-5 contains three Ig-like domains and the PDGF receptor ß contains five Ig-like domains at their extracellular regions. We showed here that the third Ig-like domain of Necl-5 cis-interacted with the fifth Ig-like domain of the PDGF receptor ß. The recombinant protein of the third Ig-like domain of Necl-5 inhibited the cis-interaction of full-length Necl-5 with the PDGF receptor ß and the PDGF-induced activation of the ERK signaling pathway that was enhanced by Necl-5. These results revealed the novel roles of the third Ig-like domain of Necl-5 and the fifth Ig-like domain of the PDGF receptor ß in its signaling.

Nectin-like molecule-4/cell adhesion molecule 4 inhibits the ligand-induced dimerization of ErbB3 with ErbB2.

The ligand-induced dimerization of cell surface single-transmembrane receptors is essential for their activation. However, physiological molecules that inhibit their dimerization and activation have not been identified. ErbB3 dimerizes with ErbB2 upon binding of heregulin (HRG) to ErbB3, causing the ErbB2-catalyzed tyrosine phosphorylation of ErbB3, which leads to the activation of the signalling pathways for cell movement and survival. Genetic disorders of this receptor cause tumorigenesis and metastasis of cancers. We show here that nectin-like molecule-4/cell adhesion molecule 4, known to serve as a tumour suppressor, interacts with ErbB3 in the absence of HRG and inhibits the HRG-induced dimerization of ErbB3 with ErbB2 and its activation. The third immunoglobulin-like domain of nectin-like molecule-4 cis-interacts with the extracellular domain 3 of ErbB3. We describe here a novel regulatory mechanism for the activation and signalling of cell surface single-transmembrane receptors.

Nectin-4 co-stimulates the prolactin receptor by interacting with SOCS1 and inhibiting its activity on the JAK2-STAT5a signaling pathway.

Cell-surface cytokine receptors are regulated by their cis-interacting stimulatory and inhibitory co-receptors. We previously showed that the Ig-like cell-adhesion molecule nectin-4 cis-interacts with the prolactin receptor through the extracellular region and stimulates prolactin-induced prolactin receptor activation and signaling, resulting in alveolar development in the mouse mammary gland. However, it remains unknown how this interaction stimulates these effects. We show here that the cis-interaction of the extracellular region of nectin-4 with the prolactin receptor was not sufficient for eliciting these effects and that the cytoplasmic region of nectin-4 was also required for this interaction. The cytoplasmic region of nectin-4 directly interacted with suppressor of cytokine signaling 1 (SOCS1), but not SOCS3, JAK2, or STAT5a, and inhibited the interaction of SOCS1 with JAK2, eventually resulting in the increased phosphorylation of STAT5a. The juxtamembrane region of nectin-4 interacted with the Src homology 2 domain of SOCS1. Both the interaction of nectin-4 with the extracellular region of the prolactin receptor and the interaction of SOCS1 with the cytoplasmic region of nectin-4 were required for the stimulatory effect of nectin-4 on the prolactin-induced prolactin receptor activation. The third Ig-like domain of nectin-4 and the second fibronectin type III domain of the prolactin receptor were involved in this cis-interaction, and both the extracellular and transmembrane regions of nectin-4 and the prolactin receptor were required for this direct interaction. These results indicate that nectin-4 serves as a stimulatory co-receptor for the prolactin receptor by regulating the feedback inhibition of SOCS1 in the JAK2-STAT5a signaling pathway.