Clathrin-binding Domain Research Articles

Metallophosphoesterase regulates light-induced rhodopsin endocytosis by promoting an association between arrestin and the adaptor protein AP2

Light-induced endocytosis of rhodopsin in the retina is critical for preventing photoreceptor hyperactivity and for the survival of photoreceptor cells. In Drosophila, this process is mediated by arrestin1 (Arr1). Because Arr1 lacks a clathrin-binding domain required for receptor internalization and the C-terminal sequence that interacts with the β-subunit of the clathrin adaptor protein AP2, the mechanism of how Arr1 mediates endocytosis of the major rhodopsin Rh1 is unclear. Here, using several approaches, including Arr binding and pulldown assays, immunofluorescence techniques, and EM imaging, we found that Drosophila metallophosphoesterase (dMPPE) is involved in light-induced rhodopsin endocytosis. We observed that the photoreceptor cells of a dmppe mutant exhibit impaired light-induced rhodopsin endocytosis and that this impairment is independent of dMPPE phosphoesterase activity. Furthermore, dMPPE directly interacted with Arr1 and promoted the association of Arr1 with AP2. Of note, genetic dmppe deletion largely prevented retinal degeneration in norpA (encoding phospholipase C) mutants, which were reported previously to contribute to retinal degeneration, by suppressing Rh1 endocytosis. Our findings demonstrate that Arr1 interacts with AP2 and that dMPPE functions as a critical regulator in Rh1 endocytosis and retinal degeneration.

DNAJ Proteins in neurodegeneration: essential and protective factors.

Maintenance of protein homeostasis is vitally important in post-mitotic cells, particularly neurons. Neurodegenerative diseases such as polyglutamine expansion disorders-like Huntington's disease or spinocerebellar ataxia (SCA), Alzheimer's disease, fronto-temporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Parkinson's disease-are often characterized by the presence of inclusions of aggregated protein. Neurons contain complex protein networks dedicated to protein quality control and maintaining protein homeostasis, or proteostasis. Molecular chaperones are a class of proteins with prominent roles in maintaining proteostasis, which act to bind and shield hydrophobic regions of nascent or misfolded proteins while allowing correct folding, conformational changes and enabling quality control. There are many different families of molecular chaperones with multiple functions in proteostasis. The DNAJ family of molecular chaperones is the largest chaperone family and is defined by the J-domain, which regulates the function of HSP70 chaperones. DNAJ proteins can also have multiple other protein domains such as ubiquitin-interacting motifs or clathrin-binding domains leading to diverse and specific roles in the cell, including targeting client proteins for degradation via the proteasome, chaperone-mediated autophagy and uncoating clathrin-coated vesicles. DNAJ proteins can also contain ER-signal peptides or mitochondrial leader sequences, targeting them to specific organelles in the cell. In this review, we discuss the multiple roles of DNAJ proteins and in particular focus on the role of DNAJ proteins in protecting against neurodegenerative diseases caused by misfolded proteins. We also discuss the role of DNAJ proteins as direct causes of inherited neurodegeneration via mutations in DNAJ family genes.This article is part of the theme issue 'Heat shock proteins as modulators and therapeutic targets of chronic disease: an integrated perspective'.

Endocytic uptake of monomeric amyloid-\u03b2 peptides is clathrin- and dynamin-independent and results in selective accumulation of A\u03b2(1\u201342) compared to A\u03b2(1\u201340)

Intraneuronal accumulation of amyloid-β (Aβ) peptides represent an early pathological feature in Alzheimer’s disease. We have therefore utilized flow cytometry and confocal microscopy in combination with endocytosis inhibition to explore the internalisation efficiency and uptake mechanisms of Aβ(1–40) and Aβ(1–42) monomers in cultured SH-SY5Y cells. We find that both variants are constitutively internalised via endocytosis and that their uptake is proportional to cellular endocytic rate. Moreover, SH-SY5Y cells internalise consistently twice the amount of Aβ(1–42) compared to Aβ(1–40); an imaging-based quantification showed that cells treated with 1 µM peptide for 8 h contained 800,000 peptides of Aβ(1–42) and 400,000 of Aβ(1–40). Both variants co-localised to >90% with lysosomes or other acidic compartments. Dynasore and chlorpromazine endocytosis inhibitors were both found to reduce uptake, particularly of Aβ(1–42). Overexpression of the C-terminal of the clathrin-binding domain of AP180, dynamin2 K44A, or Arf6 Q67L did however not reduce uptake of the Aβ variants. By contrast, perturbation of actin polymerisation and inhibition of macropinocytosis reduced Aβ(1–40) and Aβ(1–42) uptake considerably. This study clarifies mechanisms of Aβ(1–40) and Aβ(1–42) uptake, pinpoints differences between the two variants and highlights a common and putative role of macropinocytosis in the early accumulation of intraneuronal Aβ in AD.

The AP-2 complex has a specialized clathrin-independent role in apical endocytosis and polar growth in fungi.

Filamentous fungi provide excellent systems for investigating the role of the AP-2 complex in polar growth. Using Aspergillus nidulans, we show that AP-2 has a clathrin-independent essential role in polarity maintenance and growth. This is in line with a sequence analysis showing that the AP-2 β subunit (β2) of higher fungi lacks a clathrin-binding domain, and experiments showing that AP-2 does not co-localize with clathrin. We provide genetic and cellular evidence that AP-2 interacts with endocytic markers SlaBEnd4 and SagAEnd3 and the lipid flippases DnfA and DnfB in the sub-apical collar region of hyphae. The role of AP-2 in the maintenance of proper apical membrane lipid and cell wall composition is further supported by its functional interaction with BasA (sphingolipid biosynthesis) and StoA (apical sterol-rich membrane domains), and its essentiality in polar deposition of chitin. Our findings support that the AP-2 complex of dikarya has acquired, in the course of evolution, a specialized clathrin-independent function necessary for fungal polar growth.

The 4p16.3 Parkinson Disease Risk Locus Is Associated with GAK Expression and Genes Involved with the Synaptic Vesicle Membrane.

Genome-wide association studies (GWAS) have identified the GAK/DGKQ/IDUA region on 4p16.3 among the top three risk loci for Parkinson’s disease (PD), but the specific gene and risk mechanism are unclear. Here, we report transcripts containing the 3’ clathrin-binding domain of GAK identified by RNA deep-sequencing in post-mortem human brain tissue as having increased expression in PD. Furthermore, carriers of 4p16.3 PD GWAS risk SNPs show decreased expression of one of these transcripts, GAK25 (Gencode Transcript 009), which correlates with the expression of genes functioning in the synaptic vesicle membrane. Together, these findings provide strong evidence for GAK clathrin-binding- and J-domain transcripts’ influence on PD pathogenicity, and for a role for GAK in regulating synaptic function in PD.

Diverse functions of myosin VI elucidated by an isoform-specific α-helix domain.

Myosin VI functions in endocytosis and cell motility. Alternative splicing of myosin VI mRNA generates two distinct isoform types, myosin VIshort and myosin VIlong, which differ in the C-terminal region. Their physiological and pathological role remains unknown. Here we identified an isoform-specific regulatory helix, named α2-linker that defines specific conformations and hence determines the target selectivity of human myosin VI. The presence of the α2-linker structurally defines a novel clathrin-binding domain that is unique to myosin VIlong and masks the known RRL interaction motif. This finding is relevant to ovarian cancer, where alternative myosin VI splicing is aberrantly regulated, and exon skipping dictates cell addiction to myosin VIshort for tumor cell migration. The RRL interactor optineurin contributes to this process by selectively binding myosin VIshort. Thus the α2-linker acts like a molecular switch that assigns myosin VI to distinct endocytic (myosin VIlong) or migratory (myosin VIshort) functional roles.

The role of molecular chaperones in clathrin mediated vesicular trafficking.

The discovery that the 70 kD “uncoating ATPase,” which removes clathrin coats from vesicles after endocytosis, is the constitutively expressed Hsc70 chaperone was a surprise. Subsequent work, however, revealed that uncoating is an archetypal Hsp70 reaction: the cochaperone auxilin, which contains a clathrin binding domain and an Hsc70 binding J domain, recruits Hsc70*ATP to the coat and, concomitant with ATP hydrolysis, transfers it to a hydrophobic Hsc70-binding element found on a flexible tail at the C-terminus of the clathrin heavy chain. Release of clathrin in association with Hsc70*ADP follows, and the subsequent, persistent association of clathrin with Hsc70 is important to prevent aberrant clathrin polymerization. Thus, the two canonical functions of Hsp70—dissociation of existing protein complexes or aggregates, and binding to a protein to inhibit its inappropriate aggregation—are recapitulated in uncoating. Association of clathrin with Hsc70 in vivo is regulated by Hsp110, an Hsp70 NEF that is itself a member of the Hsp70 family. How Hsp110 activity is itself regulated to make Hsc70-free clathrin available for endocytosis is unclear, though at synapses it's possible that the influx of calcium that accompanies depolarization activates the Ca++/calmodulin dependent calcineurin phosphatase which then dephosphorylates and activates Hsp110 to stimulate ADP/ATP exchange and release clathrin from Hsc70*ADP:clathrin complexes.

The clathrin-binding and J-domains of GAK support the uncoating and chaperoning of clathrin by Hsc70 in the brain.

Cyclin-G-associated kinase (GAK), the ubiquitously expressed J-domain protein, is essential for the chaperoning and uncoating of clathrin that is mediated by Hsc70 (also known as HSPA8). Adjacent to the C-terminal J-domain that binds to Hsc70, GAK has a clathrin-binding domain that is linked to an N-terminal kinase domain through a PTEN-like domain. Knocking out GAK in fibroblasts caused inhibition of clathrin-dependent trafficking, which was rescued by expressing a 62-kDa fragment of GAK, comprising just the clathrin-binding and J-domains. Expressing this fragment as a transgene in mice rescued the lethality and the histological defects caused by knocking out GAK in the liver or in the brain. Furthermore, when both GAK and auxilin (also known as DNAJC6), the neuronal-specific homolog of GAK, were knocked out in the brain, mice expressing the 62-kDa GAK fragment were viable, lived a normal life-span and had no major behavior abnormalities. However, these mice were about half the size of wild-type mice. Therefore, the PTEN-like domains of GAK and auxilin are not essential for Hsc70-dependent chaperoning and uncoating of clathrin, but depending on the tissue, these domains appear to increase the efficiency of these co-chaperones.

Transport of the cholera toxin B-subunit from recycling endosomes to the Golgi requires clathrin and AP-1.

The retrograde pathway is defined by the transport of proteins and lipids from the plasma membrane through endosomes to the Golgi complex, and is essential for a variety of cellular activities. Recycling endosomes are important sorting stations for some retrograde cargo. SMAP2, a GTPase-activating protein (GAP) for Arf1 with a putative clathrin-binding domain, has previously been shown to participate in the retrograde transport of the cholera toxin B-subunit (CTxB) from recycling endosomes. Here, we found that clathrin, a vesicle coat protein, and clathrin adaptor protein complex 1 (AP-1) were present at recycling endosomes and were needed for the retrograde transport of CTxB from recycling endosomes to the Golgi, but not from the plasma membrane to recycling endosomes. SMAP2 immunoprecipitated clathrin and AP-1 through a putative clathrin-binding domain and a CALM-binding domain, and SMAP2 mutants that did not interact with clathrin or AP-1 could not localize to recycling endosomes. Moreover, knockdown of Arf1 suppressed the retrograde transport of CTxB from recycling endosomes to the Golgi. These findings suggest that retrograde transport is mediated by clathrin-coated vesicles from recycling endosomes and that the role of the coat proteins is in the recruitment of Arf GAP to transport vesicles.

The non-receptor tyrosine kinase Ack1 regulates activated EGFR fate by inducing trafficking to the p62/NBR1 pre-autophagosome

Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to the development and progression of cancer. Ack1 (activated Cdc42-associated kinase 1, also known as TNK2) is a non-receptor tyrosine kinase that has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet its precise functions remain elusive. In this report, we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon stimulation with EGF. These structures are proposed to be the isolation membranes that arise during formation of autophagosomes. In addition, we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, through a ubiquitin-associated domain, and this interaction decreases upon treatment with EGF, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, knockdown of Ack1 results in accelerated localisation of EGFR to lysosomes upon treatment with EGF. Structure-function analyses of a panel of Ack1 deletion mutants revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin-binding domain both contribute to colocalisation with EGFR, whereas the UBA domain is essential for colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.

Role of Disorder in Clathrin Lattice Assembly

Assembly of clathrin lattices is mediated by assembly/adaptor proteins which contain domains that bind lipids or membrane bound cargo proteins, and clathrin binding domains (CBDs) that recruit clathrin. Here we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins which undergo binding coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized beta turn-like structures at the two clathrin binding sites both when free and bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.

The clathrin-binding domain of CALM-AF10 alters the phenotype of myeloid neoplasms in mice

The PICALM (CALM) gene, whose product is involved in clathrin-mediated endocytosis, has been identified in two recurring chromosomal translocations, involving either MLL or MLLT10 (AF10). We developed a mouse model of CALM-AF10+ leukemia to examine the hypothesis that disruption of endocytosis contributes to leukemogenesis. Exclusion of the C-terminal portion of CALM from the fusion protein, which is required for optimal binding to clathrin, resulted in the development of a myeloproliferative disease, while inclusion of this domain led to the development of acute myeloid leukemia and changes in gene expression of several cancer-related genes, notably Pim1 and Crebbp. Nonetheless, the development of leukemia could not be attributed directly to interference with endocytosis or consequential changes in proliferation and signaling. In leukemia cells, full-length CALM-AF10 localized to the nucleus with no consistent effect on growth factor endocyctosis, and suppressed H3K79 methylation regardless of the presence of clathrin. Using FRET analysis, we show that CALM-AF10 has a propensity to homo-oligomerize, raising the possibility that the function of endocytic proteins involved in chimeric fusions may be to provide dimerization properties, a recognized mechanism for unleashing oncogenic properties of chimeric transcription factors, rather than disrupting the internalization of growth factor receptors.

The clathrin-binding domain of CALM and the OM-LZ domain of AF10 are sufficient to induce acute myeloid leukemia in mice

The t(10;11)(p13-14;q14-21) translocation, giving rise to the CALM-AF10 fusion gene, is a recurrent chromosomal rearrangement observed in patients with poor prognosis acute myeloid leukemia (AML). Although splicing of the CALM-AF10 fusion transcripts has been described in AML patients, the contribution of different CALM and AF10 domains to in vivo leukemogenesis remains to be defined. We therefore performed detailed structure-function studies of the CALM-AF10 fusion protein. We demonstrate that fusion of the C-terminal 248 amino acids of CALM, which include the clathrin-binding domain, to the octapeptide motif-leucine-zipper (OM-LZ) domain of AF10 generated a fusion protein (termed CALM-AF10 minimal fusion (MF)), with strikingly enhanced transformation capabilities in colony assays, providing an efficient system for the expeditious assessment of CALM-AF10-mediated transformation. Leukemias induced by the CALM-AF10 (MF) mutant recapitulated multiple aspects of full-length CALM-AF10-induced leukemia, including aberrant Hoxa cluster upregulation, a characteristic molecular lesion of CALM-AF10 leukemias. In summary, this study indicates that collaboration of the clathrin-binding and the OM-LZ domains of CALM-AF10 is sufficient to induce AML. These findings further suggest that future approaches to antagonize CALM-AF10-induced transformation should incorporate strategies, which aim at blocking these key domains.

Beta-Arrestin Functionally Regulates the Non-Bleaching Pigment Parapinopsin in Lamprey Pineal

The light response of vertebrate visual cells is achieved by light-sensing proteins such as opsin-based pigments as well as signal transduction proteins, including visual arrestin. Previous studies have indicated that the pineal pigment parapinopsin has evolutionally and physiologically important characteristics. Parapinopsin is phylogenetically related to vertebrate visual pigments. However, unlike the photoproduct of the visual pigment rhodopsin, which is unstable, dissociating from its chromophore and bleaching, the parapinopsin photoproduct is stable and does not release its chromophore. Here, we investigated arrestin, which regulates parapinopsin signaling, in the lamprey pineal organ, where parapinopsin and rhodopsin are localized to distinct photoreceptor cells. We found that beta-arrestin, which binds to stimulated G protein-coupled receptors (GPCRs) other than opsin-based pigments, was localized to parapinopsin-containing cells. This result stands in contrast to the localization of visual arrestin in rhodopsin-containing cells. Beta-arrestin bound to cultured cell membranes containing parapinopsin light-dependently and translocated to the outer segments of pineal parapinopsin-containing cells, suggesting that beta-arrestin binds to parapinopsin to arrest parapinopsin signaling. Interestingly, beta-arrestin colocalized with parapinopsin in the granules of the parapinopsin-expressing cell bodies under light illumination. Because beta-arrestin, which is a mediator of clathrin-mediated GPCR internalization, also served as a mediator of parapinopsin internalization in cultured cells, these results suggest that the granules were generated light-dependently by beta-arrestin-mediated internalization of parapinopsins from the outer segments. Therefore, our findings imply that beta-arrestin-mediated internalization is responsible for eliminating the stable photoproduct and restoring cell conditions to the original dark state. Taken together with a previous finding that the bleaching pigment evolved from a non-bleaching pigment, vertebrate visual arrestin may have evolved from a “beta-like” arrestin by losing its clathrin-binding domain and its function as an internalization mediator. Such changes would have followed the evolution of vertebrate visual pigments, which generate unstable photoproducts that independently decay by chromophore dissociation.

Dynamic Interactions between Clathrin and Locally Structured Elements in a Disordered Protein Mediate Clathrin Lattice Assembly

Assembly of clathrin lattices is mediated by assembly/adaptor proteins that contain domains that bind lipids or membrane-bound cargo proteins and clathrin binding domains (CBDs) that recruit clathrin. Here, we characterize the interaction between clathrin and a large fragment of the CBD of the clathrin assembly protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The locations of the two clathrin binding sites are consistent with predictions from sequence alignments of previously identified clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼ 12 degenerate repeats, each containing a single clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice.

Evolution and Origin of HRS, a Protein Interacting with Merlin, the Neurofibromatosis 2 Gene Product

Hepatocyte growth factor receptor tyrosine kinase substrate (HRS) is an endosomal protein required for trafficking receptor tyrosine kinases from the early endosome to the lysosome. HRS interacts with Merlin, the Neurofibromatosis 2 (NF2) gene product, and this interaction may be important for Merlin's tumor suppressor activity. Understanding the evolution, origin, and structure of HRS may provide new insight into Merlin function. We show that HRS homologs are present across a wide range of Metazoa with the yeast Vps27 protein as their most distant ancestor. The phylogenetic tree of the HRS family coincides with species evolution and divergence, suggesting a unique function for HRS. Sequence alignment shows that various protein domains of HRS, including the VHS domain, the FYVE domain, the UIM domain, and the clathrin-binding domain, are conserved from yeast to multicellular organisms. The evolutionary transition from unicellular to multicellular organisms was accompanied by the appearance of a binding site for Merlin, which emerges in the early Metazoa after its separation from flatworms. In addition to the region responsible for growth suppression, the Merlin-binding and STAM-binding domains of HRS are conserved among multicellular organisms. The residue equivalent to tyrosine-377, which is phosphorylated in the human HRS protein, is highly conserved throughout the HRS family. Three additional conserved boxes lacking assigned functions are found in the HRS proteins of Metazoa. While boxes 1 and 3 may constitute the Eps-15-and Snx1-binding sites, respectively, box 2, containing the residue equivalent to tyrosine-377, is likely to be important for HRS phosphorylation. While several functional domains are conserved throughout the HRS family, the STAM-binding, Merlin-binding, and growth suppression domains evolved in the early Metazoa around the time the Merlin protein emerged. As these domains appear during the transition to multicellularity, their functional roles may be related to cell-cell interaction.

The in Vivo Phosphorylation Sites in Multiple Isoforms of Amphiphysin I from Rat Brain Nerve Terminals

Amphiphysin I (amphI) is dephosphorylated by calcineurin during nerve terminal depolarization and synaptic vesicle endocytosis (SVE). Some amphI phosphorylation sites (phosphosites) have been identified with in vitro studies or phosphoproteomics screens. We used a multifaceted strategy including 32P tracking to identify all in vivo amphI phosphosites and determine their relative abundance and potential relevance to SVE. AmphI was extracted from 32P-labeled synaptosomes, phosphopeptides were isolated from proteolytic digests using TiO2 chromatography, and mass spectrometry revealed 13 sites: serines 250, 252, 262, 268, 272, 276, 285, 293, 496, 514, 539, and 626 and Thr-310. These were distributed into two clusters around the proline-rich domain and the C-terminal Src homology 3 domain. Hierarchical phosphorylation of Ser-262 preceded phosphorylation of Ser-268, -272, -276, and -285. Off-line HPLC separation and two-dimensional tryptic mapping of 32P-labeled amphI revealed that Thr-310, Ser-293, Ser-285, Ser-272, Ser-276, and Ser-268 contained the highest 32P incorporation and were the most stimulus-sensitive. Individually Thr-310 and Ser-293 were the most abundant phosphosites, incorporating 16 and 23% of the 32P. The multiple phosphopeptides containing Ser-268, Ser-276, Ser-272, and Ser-285 had 27% of the 32P. Evidence for a role for at least one proline-directed protein kinase and one non-proline-directed kinase was obtained. Four phosphosites predicted for non-proline-directed kinases, Ser-626, -250, -252, and -539, contained low amounts of 32P and were not depolarization-responsive. At least one alternatively spliced amphI isoform was identified in synaptosomes as being constitutively phosphorylated because it did not incorporate 32P during the 1-h labeling period. Multiple phosphosites from amphI-co-migrating synaptosomal proteins were also identified, including SGIP (Src homology 3 domain growth factor receptor-bound 2 (Grb2)-like (endophilin)-interacting protein 1), AAK1, eps15R, MAP6, alpha/beta-adducin, and HCN1. The results reveal two sets of amphI phosphosites that are either dynamically turning over or constitutively phosphorylated in nerve terminals and improve understanding of the role of individual amphI sites or phosphosite clusters in synaptic SVE.

β-Arrestin-Dependent Parathyroid Hormone-Stimulated Extracellular Signal-Regulated Kinase Activation and Parathyroid Hormone Type 1 Receptor Internalization

PTH regulates renal calcium homeostasis by actions on the distal nephron. PTH-induced calcium transport in mouse distal convoluted tubule (DCT) cells requires activation of ERK1/2. ERK activation by beta-adrenergic receptors occurs in a biphasic manner and involves receptor internalization. An early rapid phase is beta-arrestin (betaAr) independent, whereas prolonged activation is betaAr dependent. We characterized PTH-stimulated ERK activation and the involvement of receptor internalization and betaAr dependence. In DCT cells, PTH transiently activated ERK maximally at 5 min and then returned to baseline. betaAr dependence of PTH receptor (PTH1R)-mediated ERK stimulation was assessed using mouse embryonic fibroblasts (MEFs) from betaAr1- and -2-null mice. In wild-type MEFs, PTH(1-34)-stimulated ERK activation peaked after 5 min, was 50% maximal after 15 min, and then recovered to 80% of maximal stimulation by 30 min. In MEFs null for betaAr1 and -2, PTH-stimulated ERK activation peaked by 5 min and returned to baseline. The effect was identical in betaAr2-null MEFs. In betaAr1-null MEFs, ERK exhibited delayed activation and remained elevated. PTH-stimulated ERK activation and receptor endocytosis were not inhibited by the clathrin-binding domain of betaAr1 [Ar(319-418)]. Coexpression of the sodium proton exchanger regulatory factor 1 (NHERF1) with Ar(319-418) blocked PTH1R internalization. We conclude that PTH-stimulated ERK activation in DCT cells proceeds with a rapid but transient phase that may involve betaAr1. Furthermore, the betaAr-dependent late phase of ERK activation by PTH requires the participation of betaAr2 and PTH1R internalization.

A Novel AAK1 Splice Variant Functions at Multiple Steps of the Endocytic Pathway

Phosphorylation is a critical step in regulating receptor transport through the endocytic pathway. AAK1 is a serine/threonine kinase that is thought to coordinate the recruitment of AP-2 to receptors containing tyrosine-based internalization motifs by phosphorylating the micro2 subunit. Here we have identified a long form of AAK1 (AAK1L) that contains an extended C-terminus that encodes an additional clathrin-binding domain (CBD2) consisting of multiple low-affinity interaction motifs. Protein interaction studies demonstrate that AAK1L CBD2 directly binds clathrin. However, in vitro kinase assays reveal little difference between AAK1 isoforms in their basal or clathrin-stimulated kinase activity toward the AP-2 micro2 subunit. However, overexpression of AAK1L CBD2 impairs transferrin endocytosis, confirming an endocytic role for AAK1. Surprisingly, CBD2 overexpression or AAK1 depletion by RNA interference significantly impairs transferrin recycling from the early/sorting endosome. These observations suggest that AAK1 functions at multiple steps of the endosomal pathway by regulating transferrin internalization and its rapid recycling back to the plasma membrane from early/sorting endosome.

Dynamin 2 is required for the enhancement of HIV-1 infectivity by Nef

Nef is a virulence factor of HIV-1 and other primate lentiviruses that is crucial for rapid progression to AIDS. In cell culture, Nef increases the infectivity of HIV-1 progeny virions by an unknown mechanism. We now show that dynamin 2 (Dyn2), a key regulator of vesicular trafficking, is a binding partner of Nef that is required for its ability to increase viral infectivity. Dominant-negative Dyn2 or the depletion of Dyn2 by small interfering RNA potently inhibited the effect of Nef on HIV-1 infectivity. Furthermore, in Dyn2-depleted cells, this function of Nef could be rescued by ectopically expressed Dyn2 but not by Dyn1, a closely related isoform that does not bind Nef. The infectivity enhancement by Nef also depended on clathrin, because it was diminished in clathrin-depleted cells and profoundly inhibited in cells expressing the clathrin-binding domain of AP180, which blocks clathrin-coated pit formation but not clathrin-independent endocytosis. Together, these findings imply that the infectivity enhancement activity of Nef depends on Dyn2- and clathrin-mediated membrane invagination events.