Evidence for the interaction of alpha-actinin and calmodulin with the clathrin heavy chain

In the present study protein overlays were used to study the molecular interactions of clathrin with clathrin coat-associated proteins. Coated vesicles (CV) were isolated, subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to nitrocellulose. The transfers were quenched and equilibrated in buffer, containing 1% Triton X-100. Alpha-Actinin and calmodulin, proteins known to interact with coated vesicles, were iodinated, placed in buffer, and incubated over the transfer for 1 h. After being rinsed extensively, the total amount of 125I associated with the filters was measured, and the filters were then processed for autoradiography. For alpha-actinin, the clathrin heavy chain and a series of lower molecular weight proteins were labeled. The binding of 125I alpha-actinin was inhibited with cold ligand and selectively released from the transfer with a buffer know to strip alpha-actinin from plasma membrane preparations. For 125I calmodulin the predominant binding site was also the clathrin heavy chain. Cold ligand inhibited binding and 60% of the detectable binding were calcium dependent. In addition, when these ligands were used in competition with each other, no significant inhibition was detected in the amount of binding associated with the clathrin heavy chain. These studies show that the clathrin heavy chain is a primary site of the clathrin cage receptive to intracellular interactions and furthermore suggest that the clathrin heavy chain consists of domains of biochemical specificity which may selectively affect the activities of coated vesicles.     

Creating a chimeric clathrin heavy chain that functions independently of yeast clathrin light chain

Clathrin facilitates vesicle formation during endocytosis and sorting in the trans‐Golgi network (TGN)/endosomal system. Unlike in mammals, yeast clathrin function requires both the clathrin heavy (CHC) and clathrin light (CLC) chain, since Chc1 does not form stable trimers without Clc1. To further delineate clathrin subunit functions, we constructed a chimeric CHC protein (Chc‐YR) , which fused the N‐terminus of yeast CHC (1–1312) to the rat CHC residues 1318–1675, including the CHC trimerization region. The novel CHC‐YR allele encoded a stable protein that fractionated as a trimer. CHC‐YR also complemented chc1Δ slow growth and clathrin TGN/endosomal sorting defects. In strains depleted for Clc1 (either clc1Δ or chc1Δ clc1Δ), CHC‐YR, but not CHC1, suppressed TGN/endosomal sorting and growth phenotypes. Chc‐YR‐GFP (green fluorescent protein) localized to the TGN and cortical patches on the plasma membrane, like Chc1 and Clc1. However, Clc1‐GFP was primarily cytoplasmic in chc1Δ cells harboring pCHC‐YR, indicating that Chc‐YR does not bind yeast CLC. Still, some partial phenotypes persisted in cells with Chc‐YR, which are likely due either to loss of CLC recruitment or chimeric HC lattice instability. Ultimately, these studies have created a tool to examine non‐trimerization roles for the clathrin LC.     

Functional analysis of interaction sites on the N-terminal domain of clathrin heavy chain

In clathrin-mediated membrane traffic, clathrin does not bind directly to cargo and instead binds to adaptors that mediate this function. For endocytosis, the main adaptor is the adaptor protein (AP)-2 complex, but it is uncertain how clathrin contacts AP-2. Here we tested in human cells the importance of the three binding sites that have been identified so far on the N-terminal domain (NTD) of clathrin. We find that mutation of each of the three sites on the NTD, alone or in combination, does not block clathrin/AP-2-mediated endocytosis in the same way as deletion of the NTD. We report here the fourth and final site on the NTD that is required for clathrin/AP-2-mediated endocytic function. Each of the four interaction sites can operate alone to mediate endocytosis. The observed functional redundancy between interaction sites on the NTD explains how productivity of clathrin-coated vesicle formation is ensured.     

A monoclonal antibody to the heavy chain of clathrin.

Monoclonal antibodies have been raised to pig brain triskelions and one clone, DC41, was found to recognize the clathrin heavy chain by immunoblotting. However, both by immunofluorescence and immunoelectron microscopy, and in complete contrast to polyclonal anti-clathrin antibodies, monoclonal DC41 did not label either coated pits or coated vesicles anywhere in the cell. Instead it appeared to label the cell cytoplasm. These data suggest that DC41 recognizes a cytoplasmic form of clathrin, perhaps that form produced by uncoating of coated vesicles which is then ready to re-build another coated pit.     

In vitro, insulin receptor catalyses phosphorylation of clathrin heavy chain and a plasma membrane 180,000 molecular weight protein

Insulin receptor mutation studies that the receptor tyrosine kinase activity is necessary for receptor endocytosis, and several insulin receptor-containing tissues have a plasma membrane-associated protein (M_r 180,000, p180) whose tyrosine phosphorylation is receptor catalysed. Since clathrin heavy chain (M_r 180,000 in dodecyl sulphate gel electrophoresis) is a major component of coated vesicles, the latter functioning in receptor endocytosis, we investigated whether insulin receptors can catalyse clathrin phosphorylation and whether p180 is clathrin. Bovine brain triskelion or coated vesicles and ³²P-ATP were added to prephosphorylated insulin receptor preparations (wheat ferm agglutinin-purified human placenta membrane proteins). Antiphosphotyrosine immunoprecipitated a phosphorylated 180,000 molecular weight protein. Insulin (10⁻⁷M) increased the rate of phosphorylation. Monoclonal anti-clathrin antibody immunoprecipitated the phosphorylated 180,000 molecular weight protein, whereas monoclonal anti-insulin receptor antibodies (-IR1, MA10) immunoprecipitated both insulin receptors and the phosphorylated 180,000 molecular weight protein. In the absence of added clathrin, anticlathrin immunoprecipitated no proteins, and -IR1 imunoprecipitated only the insulin receptor. Density gradient (glycerol 7.5–30%, w/v) centrifugation separated human placenta microsomal membrane proteins into endosomal, plasma membrane, cytoplasmic and coated vesicle fractions. Antiphosphotyrosine immunoprecipitated phosphorylated-microsomal proteins that centrifugated into endosomal and plasma membrane fractions. Addition of glycerol gradient fractions to a prephosphorylated insulin receptor preparation, however, gave a tyrosine-phosphorylated 180,000 molecular weight protein when cytoplasmic and coated vesicle fractions were added. Taken together these results suggest: (1) that, in vitro, human placenta insulin receptors can phosphorylate bovine brain and human placenta clathrin heavy chain; (2) that both assembled and unassembled clathrin can be phosphorylated; and (3) that p180, the plasma membrane-associated insulin receptor substrate, is not clathrin heavy chain.     

Identification and characterization of Iporin as a novel interaction partner for rab1

Background  

The small GTPase rab1a and its isoform rab1b are essential regulating components in the vesicle transport between the ER and the Golgi apparatus. Rab1 is thought to act as a molecular switch and can change between an active GTP-bound and an inactive GDP-bound conformation. To elucidate the function of rab1, several approaches have been established to isolate effector proteins, which interact with the activated conformation of rab1. To date p115, GM130, golgin-84 and MICAL have been identified as direct interacting partners. Together with rab1, these molecules are components of a protein complex, which mediates and regulates intracellular vesicle transport.     

Light chain C-terminal region reinforces the stability of clathrin heavy chain trimers

The self-assembly of clathrin into lattices relies on the ability of heavy chain legs to form a three-legged pinwheel structure. We investigated the role of light chains in clathrin trimerization by challenging recombinant hub (plus and minus light chain) with an anionic detergent. The binding of light chain increases the amount of detergent needed to induce detrimerization, suggesting light chains reinforced hub trimers. We also show that light chain C-terminal residues are important for enhancing the in vitro assembly of hub at low pH. We assessed how much the C-terminus of light chain contributed to the stability of the trimerization domain by adding full-length and truncated light chains to trimer-defective hub mutants, C1573S and C1573A. Adding full-length LCb to C1573S caused some retrimerization, but little activity was restored, suggesting the majority of oligomeric C1573S was nonnative. A larger percentage of monomeric C1573A could be retrimerized into an assembly-competent form by adding intact LCb. We also discovered that C-terminally deleted light chains produced a heterogeneous population of hubs that were smaller than native hubs, but were assembly active. We propose a model showing how light chains reinforce the puckered clathrin triskelion. Finally, the ability of light chains to retrimerize C1573A hub suggests that the structural role of light chain may be conserved in yeast and mammals.     

Differential protein mobility of the gamma-aminobutyric acid, type A, receptor alpha and beta subunit channel-lining segments

The gamma-aminobutyric acid, type A (GABAA), receptor ion channel is lined by the second membrane-spanning (M2) segments from each of five homologous subunits that assemble to form the receptor. Gating presumably involves movement of the M2 segments. We assayed protein mobility near the M2 segment extracellular ends by measuring the ability of engineered cysteines to form disulfide bonds and high affinity Zn(2+)-binding sites. Disulfide bonds formed in alpha1beta1E270Cgamma2 but not in alpha1N275Cbeta1gamma2 or alpha1beta1gamma2K285C. Diazepam potentiation and Zn2+ inhibition demonstrated that expressed receptors contained a gamma subunit. Therefore, the disulfide bond in alpha1beta1E270Cgamma2 formed between non-adjacent subunits. In the homologous acetylcholine receptor 4-A resolution structure, the distance between alpha carbon atoms of 20' aligned positions in non-adjacent subunits is approximately 19 A. Because disulfide trapping involves covalent bond formation, it indicates the extent of movement but does not provide an indication of the energetics of protein deformation. Pairs of cysteines can form high affinity Zn(2+)-binding sites whose affinity depends on the energetics of forming a bidentate-binding site. The Zn2+ inhibition IC50 for alpha1beta1E270Cgamma2 was 34 nm. In contrast, it was greater than 100 microM in alpha1N275Cbeta1gamma2 and alpha1beta1gamma2K285C receptors. The high Zn2+ affinity in alpha1beta1E270Cgamma2 implies that this region in the beta subunit has a high protein mobility with a low energy barrier to translational motions that bring the positions into close proximity. The differential mobility of the extracellular ends of the beta and alpha M2 segments may have important implications for GABA-induced conformational changes during channel gating.     

Biochemical pharmacology of the gamma-aminobutyric acid receptor/ionophore protein

The synaptic receptor sites for the neurotransmitter gamma-aminobutyric acid (GABA) can be assayed in vitro with several radiolabeled agonists and one antagonist. Numerous criteria of specificity have been met for these binding sites. All of the ligands show heterogeneity in binding affinities. The subpopulations thus defined have a remarkably similar specificity for GABA analogs, which suggests an intimate relationship and possible interconvertibility. Modulation of GABA receptor binding by barbiturates, anions, and other membrane treatments that affect agonists and antagonists in an opposite manner suggests a three-state model of interconvertible affinities. The complex of GABA receptor and chloride ion channel contains modulatory sites for barbiturates and benzodiazepines, drugs that enhance GABA responses in neurons. The receptor complex can be solubilized in detergent with the three mutually interacting receptor activities intact. The complex has an apparent molecular weight of 355,000 and has been partially purified. GABA agonist function has been assayed at the biochemical level by measuring the activation of 36Cl- efflux from preloaded hippocampal slices by GABA, muscimol, and barbiturates. This response is blocked by the antagonists of the GABA site (bicuculline) and the barbiturate site (picrotoxin). Comparison of binding and function on the same tissue should be useful in analyzing the mechanism of action of GABA.     

Calcyon, a novel partner of clathrin light chain, stimulates clathrin-mediated endocytosis

In the central nervous system, clathrin-mediated endocytosis is crucial for efficient synaptic transmission. Clathrin-coated vesicle assembly and disassembly is regulated by some 30 adaptor and accessory proteins, most of which interact with clathrin heavy chain. Using the calcyon cytosolic domain as bait, we isolated clathrin light chain in a yeast two-hybrid screen. The interaction domain was mapped to the heavy chain binding domain and C-terminal regions of light chain. Further, the addition of the calcyon C terminus stimulated clathrin self-assembly in a dose-dependent fashion. Calcyon, which is a single transmembrane protein predominantly expressed in brain, localized to vesicular compartments within pre- and postsynaptic structures. There was a high degree of overlap in the distribution of LC and calcyon in neuronal dendrites, spines, and cell bodies. Co-immunoprecipitation studies further suggested an association of calcyon with the clathrin-mediated endocytic machinery. Compared with controls, HEK293 cells overexpressing calcyon exhibited significantly enhanced transferrin uptake but equivalent levels of recycling. Conversely, transferrin uptake was largely abolished in neocortical neurons obtained from mice homozygous for a calcyon null allele, whereas recycling proceeded at wild type levels. Collectively, these data indicate a role for calcyon in clathrin-mediated endocytosis in brain.     

Sequence of the clathrin heavy chain from Saccharomyces cerevisiae and requirement of the COOH terminus for clathrin function

The sequence of the clathrin heavy chain gene, CHC1, from Saccharomyces cerevisiae is reported. The gene encodes a protein of 1,653 amino acids that is 50% identical to the rat clathrin heavy chain (HC) (Kirchhausen, T., S. C. Harrison, E. P. Chow, R. J. Mattaliano, R. L. Ramachandran, J. Smart, and J. Brosius. 1987. Proc. Natl. Acad. Sci. USA. 84:8805-8809). The alignment extends over the complete length of the two proteins, except for a COOH-terminal extension of the rat HC and a few small gaps, primarily in the globular terminal domain. The yeast HC has four prolines in the region of the rat polypeptide that was proposed to form the binding site for clathrin light chains via an alpha-helical coiled-coil interaction. The yeast protein also lacks the COOH-terminal Pro-Gly rich segment present in the last 45 residues of the rat HC, which were proposed to be involved in the noncovalent association of HCs to form trimers at the triskelion vertex. To examine the importance of the COOH terminus of the HC for clathrin function, a HC containing a COOH-terminal deletion of 57 amino acids (HC delta 57) was expressed in clathrin-deficient yeast (chc1-delta). HC delta 57 rescued some of the phenotypes (slow growth at 30 degrees, genetic instability, and defects in mating and sporulation) associated with the chc1-delta mutation to normal or near normal. Also, truncated HCs were assembled into triskelions. However, cells with HC delta 57 were temperature sensitive for growth and still displayed a major defect in processing of the mating pheromone alpha-factor. Fewer coated vesicles could be isolated from cells with HC delta 57 than cells with the wild-type HC. This suggests that the COOH-terminal region is not required for formation of trimers, but it may be important for normal clathrin-coated vesicle structure and function.     

Identification of a residue in the gamma-aminobutyric acid type A receptor alpha subunit that differentially affects diazepam-sensitive and -insensitive benzodiazepine site binding

GABAA receptors that contain either the alpha4- or alpha6-subunit isoform do not recognize classical 1,4-benzodiazepines (BZDs). However, other classes of BZD site ligands, including beta-carbolines, bind to these diazepam-insensitive receptor subtypes. Some beta-carbolines [e.g. ethyl beta-carboline-3-carboxylate (beta-CCE) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM)] display a higher affinity for alpha4- compared to alpha6-containing receptors. In order to identify the structural determinants that underlie these affinity differences, we constructed chimeric alpha6/alpha4 subunits and co-expressed these with wild-type rat beta2 and gamma2L subunits in tsA201 cells for radioligand binding analysis. After identification of candidate regions, site-directed mutagenesis was used to narrow the ligand selectivity to a single amino acid residue (alpha6N204/alpha4I203). Substitutions at alpha6N204 did not alter the affinity of the imidazobenzodiazepine Ro15-4513. A homologous mutation in the diazepam-sensitive alpha1 subunit (S205N) resulted in a 7-8-fold reduction in affinity for the beta-carbolines examined. Although the binding of the classical agonist flunitrazepam was relatively unaffected by this mutation in the alpha1 subunit, the affinity for Ro15-1788 and Ro15-4513 was decreased by approximately 19-fold and approximately 38-fold respectively. The importance of this residue, located in the Loop C region of the extracellular N-terminus of the subunit protein, emphasizes the differential interaction of ligands with the alpha subunit in diazepam-sensitive and -insensitive receptors.     

Identification of dynein light chain 2 as an interaction partner of p21-activated kinase 1

p21-Activated kinase 1 (PAK1), a member of the evolutionarily conserved PAK family of serine/threonine kinases, is essential for a variety of cellular functions. Our previous studies showed that PAK1 participated in the apoptotic pathway mediated by p110C. To further investigate its functions, we used the yeast two-hybrid system to screen a human fetal brain cDNA library and identified dynein light chain 2 (DLC2)/myosin light chain (MLC) as an interacting partner of PAK1. The association of PAK1 with DLC2 was further confirmed by in vitro binding assay. With the stimulation of EGF, PAK1 interacted with HA-DLC2 in vivo and relocalized in cytoplasm near the perinuclear location in confocal microscope analysis. The deletion analysis showed that the interaction of DLC2 with PAK1 occurred within the residues 210-332 of PAK1. For that studies showed that DLC2 was a subunit of myosin complex, so it is possible that PAK1 binds to DLC2 and transports by myosin complex.     

Characterization of the clathrin heavy chain from Dictyostelium discoideum.

We report the cloning and analysis of a clathrin heavy-chain cDNA from the eukaryotic microorganism, Dictyostelium discoideum. A single gene, designated chcA, for the clathrin heavy chain encoded a protein of 1,694 amino acids with a molecular mass of 193,618 daltons. Comparison of the amino acid sequence with the rat and with the yeast sequence showed that the highly conserved protein was more similar to the mammalian clathrin heavy chain (57% identity) than to the yeast heavy chain (45% identity). The mRNA for the clathrin heavy chain was regulated during development. mRNA levels were highest during vegetative growth and declined as the cells progressed through the 24-hr developmental cycle. The concentration of clathrin heavy-chain protein was the same in cells grown in liquid media (high rates of pinocytosis) as in cells grown with bacteria (low rates of pinocytosis), which suggests that regulation of pinocytosis in these cells is not achieved by altering the concentration of clathrin.     

Structural elements of the gamma-aminobutyric acid type A receptor conferring subtype selectivity for benzodiazepine site ligands

gamma-aminobutyric acid type A (GABAA) receptors comprise a subfamily of ligand-gated ion channels whose activity can be modulated by ligands acting at the benzodiazepine binding site on the receptor. The benzodiazepine binding site was characterized using a site-directed mutagenesis strategy in which amino acids of the alpha5 subunit were substituted by their corresponding alpha1 residues. Given the high affinity and selectivity of alpha1-containing compared with alpha5-containing GABAA receptors for zolpidem, mutated alpha5 subunits were co-expressed with beta2 and gamma2 subunits, and the affinity of recombinant receptors for zolpidem was measured. One alpha5 mutant (bearing P162T, E200G, and T204S) exhibited properties similar to that of the alpha1 subunit, notably high affinity zolpidem binding and potentiation by zolpidem of GABA-induced chloride current. Two of these mutations, alpha5P162T and alpha5E200G, might alter binding pocket conformation, whereas alpha5T204S probably permits formation of a hydrogen bond with a proton acceptor in zolpidem. These three amino acid substitutions also influenced receptor affinity for CL218872. Our data thus suggest that corresponding amino acids of the alpha1 subunit, particularly alpha1-Ser204, are the crucial residues influencing ligand selectivity at the binding pocket of alpha1-containing receptors, and a model of this binding pocket is presented.     

Identification of a PDZ domain containing Golgi protein, GOPC, as an interaction partner of frizzled

The frizzled gene is evolutionally conserved in a wide variety of organisms including mammals, and in Drosophila, frizzled is implicated in the development of planar polarity. We describe here the isolation and characterization of a Golgi protein, GOPC, as a frizzled interacting protein. GOPC comprises one PDZ domain, two coiled-coil motifs and two evolutionally conserved regions. Immunofluorescence studies indicated that a significant fraction of GOPC protein was localized in the Golgi apparatus. Using a series of deletion mutants, we show that both coiled-coil motifs and a C-terminal conserved region were required for its Golgi localization. Interestingly, deletion mutants that lack a N-terminal conserved region or coiled-coil motifs formed aggresome-like perinuclear structure. Interaction of GOPC and frizzled was observed both in vivo and in vitro, and the PDZ domain of GOPC and the C-terminal Ser/Thr-X-Val motif of frizzled were required for their interaction. Immunofluorescence studies indicated that, although frizzled was a membrane protein, it was localized at the Golgi apparatus as well, and colocalization of GOPC and frizzled at the Golgi apparatus was observed. Furthermore, when GOPC was coexpressed with frizzled, translocation of GOPC to the plasma membrane was observed. Importantly, brefeldin A interrupted not only the localization of GOPC to the Golgi apparatus but also the translocation of frizzled to the plasma membrane, indicating that the Golgi structure was required for the proper subcellular localization of frizzled. Taken together, these results indicate that GOPC may play a role in the vesicle transport of frizzled from the Golgi apparatus to the plasma membrane.     

Establishing an ion pair interaction in the homomeric rho1 gamma-aminobutyric acid type A receptor that contributes to the gating pathway

gamma-Aminobutyric acid type A (GABA(A)) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric rho1 GABA(A) receptor. We have identified an ion pair interaction between two conserved charged residues, Glu(92) in loop 2 of the extracellular domain and Arg(258) in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily.     

Identification of cyclin D3 as a new interaction partner of lamin A/C

Lamin A/C is a major component of the nuclear lamina. An intact nuclear lamina has been proposed to be necessary for muscle differentiation. Cyclin D3 is known to be upregulated in differentiated muscle cells and to form insoluble complexes with cell-cycle regulatory factors in these cells. We have examined the possibility of direct binding interactions between lamin A/C and cyclin D3 by in vitro binding assays and co-immunoprecipitation studies with muscle cells. Our results indicate that cyclin D3 binds specifically to amino acid residues 383-474 of lamin A/C and associates with lamin A/C in muscle cells. The identification of cyclin D3 as a novel binding partner of lamin A/C has important implications for a role for lamin A/C in muscle differentiation.     

A motif in the clathrin heavy chain required for the Hsc70/auxilin uncoating reaction

The 70-kDa heat-shock cognate protein (Hsc70) chaperone is an ATP-dependent "disassembly enzyme" for many subcellular structures, including clathrin-coated vesicles where it functions as an uncoating ATPase. Hsc70, and its cochaperone auxilin together catalyze coat disassembly. Like other members of the Hsp70 chaperone family, it is thought that ATP-bound Hsc70 recognizes the clathrin triskelion through an unfolded exposed hydrophobic segment. The best candidate is the unstructured C terminus (residues 1631-1675) of the heavy chain at the foot of the tripod below the hub, containing the sequence motif QLMLT, closely related to the sequence bound preferentially by the substrate groove of Hsc70 (Fotin et al., 2004b). To test this hypothesis, we generated in insect cells recombinant mammalian triskelions that in vitro form clathrin cages and clathrin/AP-2 coats exactly like those assembled from native clathrin. We show that coats assembled from recombinant clathrin are good substrates for ATP- and auxilin-dependent, Hsc70-catalyzed uncoating. Finally, we show that this uncoating reaction proceeds normally when the coats contain recombinant heavy chains truncated C-terminal to the QLMLT motif, but very inefficiently when the motif is absent. Thus, the QLMLT motif is required for Hsc-70-facilitated uncoating, consistent with the proposal that this sequence is a specific target of the chaperone.