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PubMed2004

Agonist-specific regulation of parathyroid hormone (PTH) receptor type 2 activity: structural and functional analysis of PTH- and tuberoinfundibular peptide (TIP) 39-stimulated desensitization and internalization.

The human PTH receptor type 2 (PTH2R) is activated by PTH and tuberoinfundibular peptide of 39 residues (TIP39), resulting in cAMP and intracellular Ca signaling. We now report that, despite these similarities, PTH and TIP39 elicit distinct responses from PTH2R. First, TIP39 induced beta-arrestin and protein kinase Cbeta mobilization and receptor internalization, whereas PTH did not. However, PTH stimulated trafficking of these molecules for a chimeric PTH2R containing the N terminus and third extracellular loop of PTH receptor type 1 (PTH1R). Second, whereas PTH-stimulated cAMP activity was brief and rapidly resensitized, the response to TIP39 was sustained and partly desensitized for a prolonged period. PTH2R desensitization was mediated by beta-arrestin interaction with the C terminus (amino acids 426-457) of PTH2R, whereas beta-arrestin mobilization had a minor influence on PTH2R internalization in response to TIP39, as shown with C terminus deletion mutants and/or dominant negative forms of beta-arrestin and dynamin. These data contrast with PTH1R, at which these dominant negative mutants markedly inhibited receptor internalization. Collectively, these results further highlight how specific interactions within the ligand-receptor bimolecular complex mediate distinct postactivation responses of class II G protein- coupled receptors and provide novel insights into the physiological regulation of PTH2R activity.

Amino Acid Sequence↗
PubMed2003

IIGP1, an interferon-gamma-inducible 47-kDa GTPase of the mouse, showing cooperative enzymatic activity and GTP-dependent multimerization.

IIGP1 belongs to a well defined family of 47-kDa GTPases whose members are present at low resting levels in mouse cells but are strongly induced transcriptionally by interferons and are implicated in cell-autonomous resistance to intracellular pathogens. Recombinant IIGP1 was expressed in Escherichia coli and purified to homogeneity. Here we present a detailed biochemical characterization of IIGP1 using various biochemical and biophysical methods. IIGP1 binds to GTP and GDP with dissociation constants in the micromolar range with at least 10 times higher affinity for GDP than for GTP. IIGP1 hydrolyzes GTP to GDP, and the GTPase activity is concentration-dependent with a GTP turnover rate of 2 min-1 under saturating protein concentrations. Functional interaction between IIGP1 molecules is shown by nucleotide-dependent oligomerization in vitro. Both cooperative hydrolysis of GTP and GTP-dependent oligomerization are blocked in a mutant form of IIGP1 modified at the C terminus. IIGP1 shares micromolar nucleotide affinities and oligomerization-dependent hydrolytic activity with the 67-kDa GTPase hGBP1 (induced by type I and type II interferons), with the antiviral Mx proteins (interferon type I-induced) and with the paradigm of the self-activating large GTPases, the dynamins, with which Mx proteins show homology. The higher relative affinity for GDP and the relatively low GTPase activity distinguish IIGP1, but this study clearly adds IIGP1 and thus the p47 GTPases to the small group of cooperative GTPase families that appear to characterize the development of intracellular resistance during the interferon response to infection. The present analysis provides essential parameters to understand the molecular mechanism by which IIGP1 participates in this complex resistance program.

Animals↗
PubMed2006

Endocytic pathways regulate Toll-like receptor 4 signaling and link innate and adaptive immunity.

Immune responses are initiated when molecules of microbial origin are sensed by the Toll-like receptors (TLRs). We now report the identification of essential molecular components for the trafficking of the lipopolysaccharide (LPS) receptor complex. LPS was endocytosed by a receptor-mediated mechanism dependent on dynamin and clathrin and colocalized with TLR4 on early/sorting endosomes. TLR4 was ubiquitinated and associated with the ubiquitin-binding endosomal sorting protein hepatocyte growth factor-regulated tyrosine kinase substrate, Hrs. Inhibition of endocytosis and endosomal sorting increased LPS signaling. Finally, the LPS receptor complex was sorted to late endosomes/lysosomes for degradation and loading of associated antigens onto HLA class II molecules for presentation to CD4+ T cells. Our results show that endosomal trafficking of the LPS receptor complex is essential for signal termination and LPS-associated antigen presentation, thus controlling both innate and adaptive immunity through TLR4.

Antigen Presentation↗
PubMed1999

RACK1, a protein kinase C anchoring protein, coordinates the binding of activated protein kinase C and select pleckstrin homology domains in vitro.

The pleckstrin homology (PH) domain, identified in numerous signaling proteins including the beta-adrenergic receptor kinase (betaARK), was found to bind to various phospholipids as well as the beta subunit of heterotrimeric G proteins (Gbeta) [Touhara, K., et al. (1994) J. Biol. Chem. 269, 10217-10220]. Several PH domain-containing proteins are also substrates of protein kinase C (PKC). Because RACK1, an anchoring protein for activated PKC, is homologous to Gbeta (both contain seven repeats of the WD-40 motif), we determined (i) whether a direct interaction between various PH domains and RACK1 occurs and (ii) the effect of PKC on this interaction. We found that recombinant PH domains of several proteins exhibited differential binding to RACK1. Activated PKC and the PH domain of beta-spectrin or dynamin-1 concomitantly bound to RACK1. Although PH domains bind acidic phospholipids, the interaction between various PH domains and RACK1 was not dependent on the phospholipid activators of PKC, phosphatidylserine and 1, 2-diacylglycerol. Binding of these PH domains to RACK1 was also not affected by either inositol 1,4,5-triphosphate (IP(3)) or phosphatidylinositol 4,5-bisphosphate (PIP(2)). Our in vitro data suggest that RACK1 binds selective PH domains, and that PKC regulates this interaction. We propose that, in vivo, RACK1 may colocalize the kinase with its PH domain-containing substrates.

Animals↗
PubMed2000

Developmental expression of amphiphysin in the retinotectal system of the chick: from mRNA to protein.

The role of amphiphysin in clathrin-mediated endocytosis of synaptic vesicles is well established. However, it is still uncertain if the protein is also involved in developmental mechanisms, e.g. axon outgrowth and synapse formation. To investigate the developmental changes in the expression of amphiphysin we used the retinotectal system of the chick, a highly ordered and easily accessible primary neuronal pathway. Reverse transcription polymerase chain reaction (RT-PCR) of total RNA from chick retina and tectum revealed first transcripts for amphiphysin, dynamin and synaptotagmin at embryonic day 5 (E5) for both regions. Surprisingly, Western blots of the retina revealed an increase of protein expression for amphiphysin only after E11 in the retina and the tectum. Immunofluorescence for amphiphysin was not detectable before E10 in the developing chick retina, while other presynaptic proteins like synaptotagmin showed already intense signals in the inner and outer plexiform layers. Subsequently, amphiphysin immunoreactivity follows the expression of synaptotagmin and synaptic vesicle protein 2 (SV2) as seen in the retina and the tectum, and exhibits the same staining as the other proteins in the mature chick brain. Ultrastructural data revealed for the first time that amphiphysin is not only limited to conventional synapses but is also abundant in retinal ribbon terminals. Taken together our data reveal that: (i) there is a developmental delay between mRNA transcription and protein expression for key proteins involved in endocytosis; (ii) amphiphysin gets upregulated after synapse formation; and (iii) amphiphysin is present in the synaptic vesicle cycle in retinal ribbon synapses.

Aging↗
PubMed2001

Profilin I is essential for cell survival and cell division in early mouse development.

Profilins are thought to play a central role in the regulation of de novo actin assembly by preventing spontaneous actin polymerization through the binding of actin monomers, and the adding of monomeric actin to the barbed actin-filament ends. Other cellular functions of profilin in membrane trafficking and lipid based signaling are also likely. Binding of profilins to signaling molecules such as Arp2/3 complex, Mena, VASP, N-WASP, dynamin I, and others, further implicates profilin and actin as regulators of diverse motile activities. In mouse, two profilins are expressed from two distinct genes. Profilin I is expressed at high levels in all tissues and throughout development, whereas profilin II is expressed in neuronal cells. To examine the function of profilin I in vivo, we generated a null profilin I (pfn1(ko)) allele in mice. Homozygous pfn1(ko/ko) mice are not viable. Pfn1(ko/ko) embryos died as early as the two-cell stage, and no pfn1(ko/ko) blastocysts were detectable. Adult pfn1(ko/wt) mice show a 50% reduction in profilin I expression with no apparent impairment of cell function. However, pfn1(ko/wt) embryos have reduced survival during embryogenesis compared with wild type. Although weakly expressed in early embryos, profilin II cannot compensate for lack of profilin I. Our results indicate that mouse profilin I is an essential protein that has dosage-dependent effects on cell division and survival during embryogenesis.

Animals↗
PubMed1999

Serotonin 5-HT1A receptor-mediated Erk activation requires calcium/calmodulin-dependent receptor endocytosis.

Many receptors that couple to heterotrimeric guanine nucleotide-binding (G) proteins mediate rapid activation of the mitogen-activated protein kinases, Erk1 and Erk2. The Gi-coupled serotonin (5-hydroxytryptamine (5-HT)) 5-HT1A receptor, heterologously expressed in Chinese hamster ovary or human embryonic kidney 293 cells, mediated rapid activation of Erk1/2 via a mechanism dependent upon both Ras activation and clathrin-mediated endocytosis. This activation was attenuated by chelation of intracellular Ca2+ and Ca2+/calmodulin (CAM) inhibitors or the CAM sequestrant protein calspermin. The CAM-dependent step in the Erk1/2 activation cascade is downstream of Ras activation, because inhibitors of CAM antagonize Erk1/2 activation induced by constitutively activated mutants of Ras and c-Src but not by constitutively activated mutants of Raf and MEK (mitogen and extracellular signal-regulated kinase). Inhibitors of the classical CAM effectors myosin light chain kinase, CAM-dependent protein kinases II and IV, PP2B, and CAM-sensitive phosphodiesterase had no effect upon 5-HT1A receptor-mediated Erk1/2 activation. Because clathrin-mediated endocytosis was required for 5-HT1A receptor-mediated Erk1/2 activation, we postulated a role for CAM in receptor endocytosis. Inhibition of receptor endocytosis by use of sequestration-defective mutants of beta-arrestin1 and dynamin attenuated 5-HT1A receptor-stimulated Erk1/2 activation. Inhibition of CAM prevented agonist-dependent endocytosis of epitope-tagged 5-HT1A receptors. We conclude that CAM-dependent activation of Erk1/2 through the 5-HT1A receptor reflects its role in endocytosis of the receptor, which is a required step in the activation of MEK and subsequently Erk1/2.

Animals↗
PubMed2005

Proteomic identification of differentially expressed proteins in the aging murine olfactory system and transcriptional analysis of the associated genes.

Decline in olfactory ability has been associated with aging as well as neurodegenerative disorders. The aim of this study was to gain fundamental insight into molecular events associated with the aging olfactory system. We report a comparative proteomic analysis of the olfactory epithelium (OE) and olfactory bulb (OB) of old (80-week old) and young (6-week old) mice with further analysis of age-related differences in differentially expressed proteins at the mRNA level using real-time RT-PCR. Nine proteins in the OE and 20 in the OB were differentially expressed in old and young mice; of these, aldolase 1, peptidyl prolyl isomerase A, mitochondrial aconitase 2, mitochondrial aldehyde dehydrogenase 2 and albumin 1 were identified in the OE; and ATP synthase isoform 1, enolase 1, ferritin heavy chain, malate dehydrogenase 1, tropomyosin alpha 3 chain and dynamin 1 were identified in the OB. At the transcriptional level, aconitase 2 in the OE and ferritin heavy chain 1 in the OB were differentially expressed with aging, in concordance with the proteomic data. Our results demonstrate an altered proteomic profile of the aged murine olfactory system. The identified proteins fall into three broadly defined functional categories: (i) metabolism, (ii) transport/motility and (iii) stress response. Our transcriptional analysis provides insight into possible mechanisms by which protein expression may be regulated in the OE and OB. The results are discussed in relation to the decrement in olfactory sensitivity with aging.

Age Factors↗
PubMed1999

Molecular mechanisms of angiotensin II receptor internalization.

Angiotensin II (AngII) initiates cellular responses by activation of type I (AT1) and type 2 (AT2) angiotensin receptors. Both AT1 and AT1 receptors have seven transmembrane structures characteristic of G protein-coupled receptors, but only the AT1 receptor undergoes rapid internalization upon agonist binding. In addition to the agonist hormone, the peptide antagonist [Sar1,Ile8]AngII can also induce internalization of the AT1a receptor expressed in mammalian cell lines, but the nonpeptide AT1 receptor blocker losartan does not internalize. AT1 receptor internalization occurs via clathrin-coated pits, but there is evidence that, in contrast to the internalization of other G protein-coupled receptors, the internalization of the AT1 receptor is independent of dynamin and beta-arrestin. Mutagenesis studies demonstrated that AT1 receptor internalization requires two regions in the cytoplasmic tail of the receptor, but it is independent of G protein activation. The dependence of AT1 receptor internalization on the presence of a serine-threonine-rich region suggests that phosphorylation of the receptor tail may regulate the internalization process. The possible role of AT1 receptor internalization in sustained signal generation has been suggested, but its relationship to nuclear AngII receptors is not completely understood.

Amino Acid Sequence↗
PubMed1998

A dynamin-like protein in Arabidopsis thaliana is involved in biogenesis of thylakoid membranes.

Dynamin, a GTP-binding protein found in rat brain, plays a role in endocytosis. Suborganellar fractionation studies of Arabidopsis leaf tissue revealed that a dynamin-like protein, ADL1, is localized in the thylakoid membranes of chloroplasts. This notion was supported further by in vivo targeting experiments using an ADL1-green fluorescent fusion protein and immunogold labeling with the anti-ADL1 antibody. Transgenic plants harboring various deletion mutant genes of ADL1 had a yellow leaf phenotype where the cells had very few chloroplasts. In addition, the remaining chloroplasts appeared morphologically not fully developed. The detailed structure of the chloroplasts revealed by electron microscopy showed a greatly reduced amount of thylakoid membranes. Also, the level of thylakoid membrane proteins such as the light-harvesting complex II and CP29 was greatly reduced in these transgenic plants. When we examined the expression of the ADL1 deletion mutant genes, these genes were highly expressed at the transcriptional level. However, the mutant ADL1s were not detectable at the protein level by Western blot analysis. Moreover, the endogenous ADL1 protein level was greatly reduced in these transgenic plants, probably due to a post-transcriptional silencing effect of the transgenes. We propose, therefore, that ADLl is involved in the biogenesis of thylakoid membranes.

Arabidopsis↗
PubMed2001

Transforming growth factor (TGF)-beta 1 internalization: modulation by ligand interaction with TGF-beta receptors types I and II and a mechanism that is distinct from clathrin-mediated endocytosis.

Transforming growth factor-beta (TGF-beta) internalization was studied by monitoring the uptake of (125)I-TGF-beta1 in Mv1Lu cells, which endogenously express TGF-beta receptors types I (RI), II (RII), and III (RIII), and 293 cells transfected with RI and RII. At 37 degrees C internalization occurred rapidly, within 10 min of ligand addition. Internalization was optimal in 293 cells expressing both RI and RII. Internalization was prevented by phenylarsine oxide, a nonspecific inhibitor of receptor internalization, but was not affected by reagents that interfere with clathrin-mediated endocytosis such as monodansylcadaverine, K44A dynamin, and inhibitors of endosomal acidification. Electron microscopic examination of Mv1Lu cells treated with (125)I- TGF-beta1 at 37 degrees C indicated that internalization occurred via a noncoated vesicular mechanism. Internalization was prevented by prebinding cells with TGF-beta1 at 4 degrees C for 2 h prior to switching the cells to 37 degrees C. This was attributed to a loss of receptor binding, as indicated by a rapid decrease in the amount of TGF-beta1 bound to the cell surface at 37 degrees C and by a reduction in the labeling intensities of RI and RII in (125)I-TGF-beta1-cross-linking experiments. Mv1Lu or 293 (RI+RII) cells, prebound with TGF-beta1 at 4 degrees C and subsequently stripped of ligand by an acid wash, nevertheless initiated a signaling response upon transfer to 37 degrees C, suggesting that prebinding promotes formation of stable RI.RII complexes that can signal independently of ligand.

Animals↗
PubMed2006

Antagonistic regulation of ROMK by long and kidney-specific WNK1 isoforms.

WNK kinases are serine-threonine kinases with an atypical placement of the catalytic lysine. Intronic deletions with increased expression of a ubiquitous long WNK1 transcript cause pseudohypoaldosteronism type 2 (PHA II), characterized by hypertension and hyperkalemia. Here, we report that long WNK1 inhibited ROMK1 by stimulating its endocytosis. Inhibition of ROMK by long WNK1 was synergistic with, but not dependent on, WNK4. A smaller transcript of WNK1 lacking the N-terminal 1-437 amino acids is expressed highly in the kidney. Whether expression of the KS-WNK1 (kidney-specific, KS) is altered in PHA II is not known. We found that KS-WNK1 did not inhibit ROMK1 but reversed the inhibition of ROMK1 caused by long WNK1. Consistent with the lack of inhibition by KS-WNK1, we found that amino acids 1-491 of the long WNK1 were sufficient for inhibiting ROMK. Dietary K(+) restriction decreases ROMK abundance in the renal cortical-collecting ducts by stimulating endocytosis, an adaptative response important for conservation of K(+) during K(+) deficiency. We found that K(+) restriction in rats increased whole-kidney transcript of long WNK1 while decreasing that of KS-WNK1. Thus, KS-WNK1 is a physiological antagonist of long WNK1. Hyperkalemia in PHA II patients with PHA II mutations may be caused, at least partially, by increased expression of long WNK1 with or without decreased expression of KS-WNK1.

Amino Acid Sequence↗
PubMed2005

Initial entry of IRAP into the insulin-responsive storage compartment occurs prior to basal or insulin-stimulated plasma membrane recycling.

To examine the acquisition of insulin sensitivity after the initial biosynthesis of the insulin-responsive aminopeptidase (IRAP), 3T3-L1 adipocytes were transfected with an enhanced green fluorescent protein-IRAP (EGFP-IRAP) fusion protein. In the absence of insulin, IRAP was rapidly localized (1-3 h) to secretory membranes and retained in these intracellular membrane compartments with little accumulation at the plasma membrane. However, insulin was unable to induce translocation to the plasma membrane until 6-9 h after biosynthesis. This was in marked contrast to another type II membrane protein (syntaxin 3) that rapidly defaulted to the plasma membrane 3 h after expression. In parallel with the time-dependent acquisition of insulin responsiveness, the newly synthesized IRAP protein converted from a brefeldin A-sensitive to a brefeldin A-insensitive state. The initial trafficking of IRAP to the insulin-responsive compartment was independent of plasma membrane endocytosis, as expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis but had no effect on the insulin-stimulated translocation of the newly synthesized IRAP protein.

3T3-L1 Cells↗
PubMed2004

Protein kinase C mediates phosphorylation, desensitization, and trafficking of the D2 dopamine receptor.

Previously, D2 dopamine receptors (D2 DARs) have been shown to undergo G-protein-coupled receptor kinase phosphorylation in an agonist-specific fashion. We have now investigated the ability of the second messenger-activated protein kinases, protein kinase A (PKA) and protein kinase C (PKC), to mediate phosphorylation and desensitization of the D2 DAR. HEK293T cells were transiently transfected with the D2 DAR and then treated with intracellular activators and inhibitors of PKA or PKC. Treatment with agents that increase cAMP, and activate PKA, had no effect on the phosphorylation state of the D2 DAR, suggesting that PKA does not phosphorylate the D2 DAR in HEK293T cells. In contrast, cellular treatment with phorbol 12-myristate 13-acetate (PMA), a PKC activator, resulted in an approximately 3-fold increase in D2 DAR phosphorylation. The phosphorylation was specific for PKC as the PMA effect was mimicked by phorbol 12,13-dibutyrate, but not by 4alpha-phorbol 12,13-didecanoate, active and inactive, phorbol diesters, respectively. The PMA-mediated D2 DAR phosphorylation was completely blocked by co-treatment with the PKC inhibitor, bisindolylmaleimide II, and augmented by co-transfection with PKCbetaI. In contrast, PKC inhibition had no effect on agonist-promoted phosphorylation, suggesting that PKC is not involved in this response. PKC phosphorylation of the D2 DAR was found to promote receptor desensitization as reflected by a decrease in agonist potency for inhibiting cAMP accumulation. Most interestingly, PKC phosphorylation also promoted internalization of the D2 DAR through a beta-arrestin- and dynamin-dependent pathway, a response not usually associated with PKC phosphorylation of G-protein-coupled receptors. Site-directed mutagenesis experiments resulted in the identification of two domains of PKC phosphorylation sites within the third intracellular loop of the receptor. Both of these domains are involved in regulating sequestration of the D2 DAR, whereas only one domain is involved in receptor desensitization. These results indicate that PKC can mediate phosphorylation of the D2 DAR, resulting in both functional desensitization and receptor internalization.

Amino Acid Sequence↗
PubMed2006

Studying the protein organization of the postsynaptic density by a novel solid phase- and chemical cross-linking-based technology.

Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules with their original abundances could provide information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslips. By using these beads, we determined the interior or superficial locations of five major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian central nervous system. The results indicate that alpha,beta-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, an hsp70 protein, alpha-internexin, dynamin, and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, kainate receptors, N-cadherin, beta-catenin, N-ethylmaleimide-sensitive factor, an hsc70 protein, and actin reside on the surface of the PSD. The results further suggest that the N-methyl-d-aspartate receptors and the alpha-subunits of calcium/calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD although with unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.

Animals↗
PubMed1999

The structural era of endocytosis.

Endocytosis is crucial for an array of cellular functions and can occur through several distinct mechanisms with the capacity to internalize anything from small molecules to entire cells. The clathrin-mediated endocytic pathway has recently received considerable attention because of (i) the identification of an array of molecules that orchestrate the assembly of clathrin-coated vesicles and the selection of the vesicle cargo and (ii) the resolution of structures for a number of these proteins. Together, these data provide an initial three-dimensional framework for understanding the clathrin endocytic machinery.

Animals↗
PubMed2004

Identification of residues in the human guanylate-binding protein 1 critical for nucleotide binding and cooperative GTP hydrolysis.

The guanylate-binding proteins (GBPs) form a group of interferon-gamma inducible GTP-binding proteins which belong to the family of dynamin-related proteins. Like other members of this family, human guanylate-binding protein 1 (hGBP1) shows nucleotide-dependent oligomerisation that stimulates the GTPase activity of the protein. A unique feature of the GBPs is their ability to hydrolyse GTP to GDP and GMP. In order to elucidate the relationship between these findings, we designed point mutants in the phosphate-binding loop (P-loop) as well as in the switch I and switch II regions of the protein based on the crystal structure of hGBP1. These mutant proteins were analysed for their interaction with guanine nucleotides labeled with a fluorescence dye and for their ability to hydrolyse GTP in a cooperative manner. We identified mutations of amino acid residues that decrease GTPase activity by orders of magnitude a part of which are conserved in GTP-binding proteins. In addition, mutants in the P-loop were characterized that strongly impair binding of nucleotide. In consequence, together with altered GTPase activity and given cellular nucleotide concentrations this results in hGBP1 mutants prevailingly resting in the nucleotide-free (K51A and S52N) or the GTP bound form (R48A), respectively. Using size-exclusion chromatography and analytical ultracentrifugation we addressed the impact on protein oligomerisation. In summary, mutants of hGBP1 were identified and biochemically characterized providing hGBP1 locked in defined states in order to investigate their functional role in future cell biology studies.

Amino Acid Sequence↗
PubMed1992

The postsynaptic density: constituent and associated proteins characterized by electrophoresis, immunoblotting, and peptide sequencing.

The proteins of the postsynaptic density (PSD) fraction of cerebral cortex were resolved by two-dimensional electrophoresis (2DE) and more than 30 proteins identified by characteristic 2DE mobility, immunoblotting with specific antibodies, and N-terminal and peptide sequencing. The PSD fraction is enriched for spectrin, actin, tublin and microtubule associated protein II, myosin, enzymes of glycolysis, creatine kinase, elongation factor 1 alpha, and receptor protein. The three neurofilament proteins are detected but a 58-kDa protein is prominent and is, by peptide sequencing, the bovine homolog of the recently cloned 66-kDa neurofilament protein; in contrast to the latter, however, it is enriched in cerebrum compared with spinal cord. A 68-kDa protein is identified as a member of the hsp70/BiP family of proteins. A protein, designated dynamin, indicating its putative role as a microtubule motor, is identified as a major protein, is found, however, greatly enriched in the particulate fraction, and is significantly denaturant and detergent insoluble. A protein designated N-ethylmaleimide-sensitive factor is also detected. Thus, two proteins implicated in vesicular transport are present in the PSD fraction. Seven polyclonal antibodies were produced to 2DE separated and electroeluted proteins of the PSD and were identified by peptide sequence analysis and 2DE profile as the hsp70/BiP homologous protein, the novel neurofilament protein synapsin IIa, pyruvate kinase, dynamin, aconitase and an unknown contaminating protein, and a 115-kDa protein that by subcellular fractionation and immunoblotting is a diagnostic PSD molecule. In addition, peptide sequences are obtained for four additional higher molecular weight proteins of the PSD that are not related at the level of primary structure to any known proteins.

Actins↗