Misfolding of fukutin-related protein (FKRP) variants in congenital and limb girdle muscular dystrophies.

Fukutin-related protein (FKRP, MIM ID 606596) variants cause a range of muscular dystrophies associated with hypo-glycosylation of the matrix receptor, α-dystroglycan. These disorders are almost exclusively caused by homozygous or compound heterozygous missense variants in the FKRP gene that encodes a ribitol phosphotransferase. To understand how seemingly diverse FKRP missense mutations may contribute to disease, we examined the synthesis, intracellular dynamics, and structural consequences of a panel of missense mutations that encompass the disease spectrum. Under non-reducing electrophoresis conditions, wild type FKRP appears to be monomeric whereas disease-causing FKRP mutants migrate as high molecular weight, disulfide-bonded aggregates. These results were recapitulated using cysteine-scanning mutagenesis suggesting that abnormal disulfide bonding may perturb FKRP folding. Using fluorescence recovery after photobleaching, we found that the intracellular mobility of most FKRP mutants in ATP-depleted cells is dramatically reduced but can, in most cases, be rescued with reducing agents. Mass spectrometry showed that wild type and mutant FKRP differentially associate with several endoplasmic reticulum (ER)-resident chaperones. Finally, structural modelling revealed that disease-associated FKRP missense variants affected the local environment of the protein in small but significant ways. These data demonstrate that protein misfolding contributes to the molecular pathophysiology of FKRP-deficient muscular dystrophies and suggest that molecules that rescue this folding defect could be used to treat these disorders.

Identification of a novel region of the GABA(B2) C-terminus that regulates surface expression and neuronal targeting of the GABA(B) receptor.

GABA(B) is a G protein-coupled receptor composed of two subunits, GABA(B1) and GABA(B2). GABA(B1) contains an endoplasmic reticulum-retention sequence and is trafficked to the cell surface only in association with GABA(B2). To determine whether the C-terminus of GABA(B2) regulates GABA(B) trafficking, we constructed forms of GABA(B2) with various C-terminal truncations and examined their surface expression. Truncation of GABA(B2) after residue 841 significantly reduced surface expression of both the subunit and the heterodimerized receptor. Turnover of the Delta841 construct, however, did not differ from that of full-length GABA(B2). To determine whether the C-terminus of GABA(B2) might target GABA(B) to neurites, cultured hippocampal neurons were transfected with the truncated GABA(B2) constructs. Truncation of GABA(B2) at residue 841 resulted in primarily somatic localization; furthermore, axonal trafficking of this construct was significantly more restricted than dendritic trafficking. Finally, to biochemically assess trafficking of the truncated GABA(B2) constructs, we digested transfected HEK293 cell lysates with endoglycosidase H. When GABA(B2) was truncated at residue 841, it became sensitive to digestion by this enzyme, indicating incomplete trafficking. Taken together, these data show that the region of the GABA(B2) C-terminus between residues 841 and 862 is important for regulating forward trafficking and neuronal targeting of the GABA(B) receptor.

The fats of life: the importance and function of protein acylation.

Interest in the study of the direct attachment of fatty acids to cellular proteins, termed protein acylation, has been greatly stimulated by recent experimentation that has increased our understanding of the function of the attached lipid. These developments are described, and the possibility that inhibitors of protein acylation might provide new drugs is discussed.

The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.

An antiserum to mGluR1 alpha labeled a 160 kd protein in immunoblots of membranes derived from rat brain or cells transfected with mGluR1 alpha. Immunoreactivity for mGluR1 alpha was present in discrete subpopulations of neurons. The GABAergic neurons of the cerebellar cortex were strongly immunoreactive; only some Golgi cells were immunonegative. Somatostatin/GABA-immunopositive cells in the neocortex and hippocampus were enriched in mGluR1 alpha. The hippocampal cells had spiny dendrites that were precisely codistributed with the local axon collaterals of pyramidal and granule cells. Electron microscopic immunometal detection of mGluR1 alpha showed a preferential localization at the periphery of the extensive postsynaptic densities of type 1 synapses in both the cerebellum and the hippocampus. The receptor was also present at sites in the dendritic and somatic membrane where synapses were not located.

The C-terminus of the metabotropic glutamate receptor 1b regulates dimerization of the receptor.

The Group C G protein-coupled receptors include the metabotropic glutamate receptors (mGluRs), the GABA(B) receptor, the calcium sensor and several taste receptors, most of which are obligate dimers, indeed recent work has shown that dimerization is necessary for the activation of these receptors. Consequently factors that regulate their ability to homo- or heterodimerize are important. The Group 1 mGluRs include mGluR1 and mGluR5 both of which have splice variants with altered C-termini. In this study, we show that mGluR1b is a dimer and that it does not efficiently heterodimerize with mGluR1a, unlike the two splice variants of mGluR5 that can heterodimerize. Mutation of a positively charged motif (RRKK) at the C-terminus of the mGluR1b tail permits mGluR1b to heterodimerize with mGluR1a. Co-expression of mGluR1a and mGluR1b in COS-7 cells results in the accumulation of mGluR1b in intracellular inclusions that do not contain mGluR1a. This behaviour is mimicked by a chimera of the lymphocyte antigen CD2 with the C-terminus of mGluR1b (pCD1b) and depends on the presence of the RRKK motif. These accumulations are immunoreactive for endoplasmic reticulum (ER) markers, but not Golgi and ERGIC markers. This segregation of mGluR1b from other ER proteins may contribute to its failure to dimerize with mGluR1a.

Monoclonal antibodies raised against cell membrane components of human bladder tumor tissue recognizing subpopulations in normal urothelium.

Monoclonal antibodies to human bladder carcinoma membrane antigens were produced by fusion of MOPC-21 NS/1 mouse myeloma cells with spleen cells from BALB/c mice immunized against a crude membrane extract from a metastatic bladder carcinoma. Hybrids were screened for antibody production in a solid-phase radioimmunoassay and selected for their reactivity with subpopulations of urothelial cells on normal bladder tissue sections. Three antibody groups were defined: Group I (4-72-2) was urothelium specific and stained the basal and intermediate cells in normal urothelium; group II (3-48-2, 48-1, and 3-50-3) showed reactivity with intermediate and superficial cells; group III (8-30-3, 77-1, 2-94-2, 3-71-1, and 94-3) was restricted to antigens on the luminal membrane of superficial cells. All antibodies recognized antigenic determinants in fixed paraffin-embedded material and within groups showed a range of staining patterns in other tissues. Studies on sections representing different stages of neoplastic progression showed disruption in the antibody-staining pattern in urothelium and, in all cases, a strong distinct staining of invasive tumor areas and metastatic secondary tumors. Biochemical analysis of the antigens defined at least three antigenic systems, two of which consisted of molecules having Mr of 250,000 and 300,000 as judged by Western blot analysis. Antigenic determinants recognized by some antibodies (3-48-2, 48-1, 3-50-3, 8-30-3, 77-1, and 3-71-1) were shown to be carbohydrate by reactivity with glycolipid fraction and suggest that antibodies within groups recognize different epitopes on the same molecule.

Characterization of the fibronectin synthesized by human germ cell tumors.

The ability of the human germ cell tumor cell lines Tera 1, Tera 2, PA-1, LICR LON HT 39/7, LICR LON HT3B1, and LICR LON HT 53 to synthesize and secrete fibronectin has been studied. The presence of cellular fibronectin was examined using indirect immunofluorescence, whereas the synthesis and secretion of the protein were studied using specific immunoprecipitation from cultures radioactively labeled with [35S]methionine. Two of the cell lines, LICR LON HT 39/7 and Tera 1, did not synthesize fibronectin, whereas all the other cell lines did. Plasma membrane fibronectin could not be demonstrated on any of the cell lines, although cytoplasmic fibronectin was easily demonstrable. The cells appear therefore to synthesize fibronectin but not retain it. Sodium dodecyl sulfate:polyacrylamide gel electrophoresis of the secreted fibronectin produced by the human teratoma cell lines showed that it had an apparent molecular weight greater than that produced by adult human breast fibroblasts or human plasma fibronectin. Peptide mapping of this secreted germ cell tumor fibronectin, by partial proteolytic cleavage, yielded peptide patterns similar to those obtained from either human plasma fibronectin or adult human breast fibroblast fibronectin. The difference in molecular weight between the fibronectins may therefore be due to changes in their patterns of glycosylation.

Immunohistochemical study of beta- and kappa-casein in the human breast and breast carcinomas, using monoclonal antibodies.

The immunohistochemical analysis of human beta- and kappa-caseins in the human breast, benign breast disease, and breast carcinomas is reported. The monoclonal antibodies LICR-LON-32.2 and LICR-LON-14.1 which react with human beta- and kappa-casein, respectively (Earl, H. M., and McIlhinney, R. A. J. Mol. Immunol., 22: 981-991, 1985) were used for these studies. Human beta- and kappa-caseins were detected in lactating human breast tissue, lactational foci in the resting breast, and heterogeneously in the 16th-week pregnant breast, but not in normal breast tissue. In benign breast disease occasional epithelial cells were demonstrated to synthesize beta- and kappa-caseins, but this finding did not appear to correlate with the hormonal status or previous obstetric histories of the patients. However, similar studies in 45 breast carcinomas with a wide range of estrogen receptor content, demonstrated no detectable beta- or kappa-casein. These results demonstrate that the caseins, which are biochemical markers of mammary gland differentiated function, and have been previously put forward as markers of breast cancer, were not synthesized in the 45 human breast carcinomas studied here, even in tumors with high levels of estrogen receptor protein.

Palmitoylation of endogenous and viral acceptor proteins by fatty acyltransferase (PAT) present in erythrocyte ghosts and in placental membranes.

Human erythrocyte ghosts were shown to have palmitoylating activity which acylates both endogenous ghost polypeptides and exogenous proteins derived from Semliki Forest virus (SFV). Cell-free fatty acid transfer from [3H]palmitoyl-CoA to endogenous protein was greatly enhanced in ghosts when pre-existing fatty acids linked to the endogenous acyl proteins were removed by hydroxylamine treatment prior to the transfer reaction. In contrast to erythrocyte acyl proteins acceptor proteins present in human placental membranes were palmitoylated in vitro to a similar extent with or without prior deacylation by hydroxylamine treatment. This indicates the presence of large pools of non-acylated proteins in placenta and small pools in erythrocytes. In testing for the protein substrate specificity of the palmitoyl transferase (PAT) present in ghosts we found that the SFV acceptor proteins, which are totally unrelated to erythrocytes, competed with the palmitoylation of endogenous ghost protein acceptors. This palmitoylating enzyme is inhibited by Cibacron Blue, SDS, and heat treatment, but stimulated in the presence of low concentrations of mild detergent (TX-100). Since PAT operating at the surface membrane of red blood cells has properties very similar to those of PAT present in human placental microsomes [1], we suggest that only one type of PAT may transfer fatty acids to various acylproteins that occur at multiple locations in different tissues [2].

Identification, cloning and analysis of expression of a new alternatively spliced form of the metabotropic glutamate receptor mGluR1 mRNA1.

We have applied quantitative RT-PCR analysis to characterise relative levels of expression of the alternatively spliced mGluR1 mRNAs. This has also allowed us to identify and clone a new alternatively spliced form of the mGluR1 mRNA. The newly identified mGluR1f mRNA is expressed at moderate levels in rat brain, reaching its maximum in cortex. mGluR1f differs from the mGluR1a mRNA by deletion of a 35-bp fragment of the mGluR1a/alpha coding sequence and insertion of an 85-bp fragment, found only in mGluR1b/beta mRNA.

Consequences of lipid raft association on G-protein-coupled receptor function.

GPCRs (G-protein-coupled receptors) play key roles in many cellular processes, and malfunction may lead to a range of pathologies, including psychiatric and neurological disorders. It is therefore not surprising that this group of receptors supplies a majority of the targets for pharmaceutical drug development. Despite their importance, the mechanisms that regulate their function and signalling still remain only partially understood. Recently, it has become evident that a subset of GPCRs is not homogeneously distributed in the plasma membrane, but localizes instead to specific membrane microdomains known as lipid rafts. Lipid rafts are characterized by their enrichment in cholesterol and sphingolipids, and have been suggested to serve as platforms for a range of cellular signalling complexes. In the present review, we will be discussing the effects of the lipid raft environment on trafficking, signalling and internalization of raft-associated GPCRs.

Molecular characterisation of two structurally distinct groups of human homers, generated by extensive alternative splicing.

Homer proteins bind specifically to the C termini of the metabotropic glutamate receptor mGluR1alpha/a and mGluR5, play a role in their targeting and modulate their synaptic properties. We have discovered that extensive alternative splicing generates a family of 17 Homer proteins. These fall into two distinct groups of 12 "long" Homers, which all have a coiled-coil domain at their C termini, and five "short" Homers, which lack such a domain. All Homers contain the N-terminal sequence responsible for their binding to mGluR1alpha/a receptors and can be co-localised with the recombinantly expressed mGluR1alpha/a protein in HEK-293 cells. The existence of the long and the short variants of each of the Homer-1, Homer-2 and Homer-3 proteins reflects the fundamental principles of Homer functions.

Monoclonal antibodies to human casein.

Two monoclonal antibodies, LICR-LON-32.2 (32.2) and LICR-LON-14.1 (14.1), are described which react with human casein. 32.2 reacts with human beta-casein and 14.1 with human kappa-casein. 32.2 also reacts with rat band 2 casein and bovine beta-casein, but 14.1 appears to be specific for human kappa-casein. These monoclonal antibodies do not cross-react with other milk proteins.

A marker of human foetal endoderm defined by a monoclonal antibody involves Type 1 blood group chains.

This report demonstrates that a marker of human embryonic endoderm and embryonal carcinoma cells recognized by a hybridoma antibody FC 10.2, involves Type 1 blood group chains with the sequence Gal beta 1 leads to 3G1cNAc beta 1 leads to 3Gal beta 1 leads to 4G1c. This conclusion has been reached from antigenic analyses of meconium, ovarian cyst glycoproteins, oligosaccharides and glycolipids having Type 1 or Type 2 blood group chains. From knowledge of saccharide sequences and blood group related antigens in gastrointestinal tissues of man, we deduce that the 'disappearance' of FC 10.2 antigen from the normal, differentiated cells of the adult may result from masking by additional glycosylations or other substitutions.

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres.

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.

Interactions of the C Terminus of Metabotropic Glutamate Receptor Type 1α with Rat Brain Proteins: Evidence for a Direct Interaction with Tubulin.

Metabotropic glutamate receptors (mGluRs) are coupled to G protein second messenger pathways and modulate glutamate neurotransmission in the brain, where they are targeted to specific synaptic locations. As part of a strategy for defining the mechanisms for the specific targeting of mGluR1 α, rat brain proteins which interact with the intracellular carboxy terminus of mGluR1 α have been characterized, using affinity chromatography on a glutathione S-transferase fusion protein that contains the last 86 amino acids of mGluR1 α. Three of the proteins specifically eluted from the affinity column yielded protein sequences, two of which were identified as glyceraldehyde-3-phosphate dehydrogenase and ß-tubulin ; the other was an unknown protein. The identity of tubulin was confirmed by western immunoblotting. Using a solid-phase binding assay, the mGluR1 α-tubulin interaction was shown to be direct, specific, and saturable with a KD of 2.3 ± 0.4 µM. In addition, mGluR1 α, but not mGluR2/3 or mGluR4, could be coimmunoprecipitated from solubilized brain extracts with tubulin using anti-ß-tubulin antibodies. However, mGluR1 α could not be coimmunoprecipitated with the tubulin binding protein gephyrin, nor could it be coimmunoprecipitated with PSD95. Collectively these data demonstrate that the last 86 amino acids of the carboxyl-terminal tail of mGluR1 α are sufficient to determine its interaction with tubulin and that there is an association of this receptor with tubulin in rat brain.

Differential internalisation of mGluR1 splice variants in response to agonist and phorbol esters in permanently transfected BHK cells.

The internalisation of metabotropic glutamate receptor (mGluR1alpha) and its splice variant (mGluR1beta), in response to agonist and phorbol esters (PMA), has been studied. Both mGluR1alpha and mGluR1beta exhibit a similar rate of internalisation following PMA treatment, with a shift in their distribution from plasma membrane to endosome-enriched membrane fractions. Agonist challenge however caused a rapid loss, within 5-10 min, of mGluR1beta but not mGluR1alpha from the cell surface. These results show that the two forms of mGluR1 show different internalisation responses to agonist and suggest that the C-terminal region of the molecule plays an important role in this phenomenon.

Rapid agonist mediated phosphorylation of the metabotropic glutamate receptor 1 alpha by protein kinase C in permanently transfected BHK cells.

Clonal BHK cells permanently transfected with the metabotropic glutamate receptor 1 alpha (mGluR1 alpha), which is coupled to phospholipase C, were used to study the phosphorylation state of the receptor. Cells were labelled with 32PO4(3-), lysed, the receptor immunoprecipitated with specific anti-peptide antibodies and the immunoprecipitates analysed by SDS-PAGE followed by autoradiography. A significant basal level of receptor phosphorylation was observed which was rapidly and transiently increased in response to agonist activation of the receptor. This agonist effect was found to be dose dependent with a rapid time course and could be abolished by the specific PKC inhibitor Ro318220, suggesting that PKC was responsible for the agonist mediated phosphorylation of the receptor.

Cell surface expression of the metabotropic glutamate receptor type 1alpha is regulated by the C-terminal tail.

The cell surface expression of metabotropic glutamate receptor type I splice variants has been studied using cell surface biotinylation. Co-expression of the last 86 residues of the C-terminal tail of mGluR1alpha (F2-protein) with mGluR1alpha caused a significant reduction of the amount of the cell surface receptor when compared to that in cells transfected with mGlur1alpha alone, and this was accompanied by a reduction in the production of inositol following agonist stimulation of the cells. In contrast, cell surface expression of mGluR1beta was unaltered by co-expression with the F2-protein. These results suggest that the C-terminal tail of mGluR1alpha regulates cell surface expression of the receptor.

Functional regulation of metabotropic glutamate receptor type 1c: a role for phosphorylation in the desensitization of the receptor.

The phosphorylation and desensitization of metabotropic glutamate receptor type 1c in response to agonist and phorbol esters has been studied. Specific immunoprecipitation of mGluR1c from cells treated with agonist or PMA showed a time-dependent increase in the phosphorylation of a membrane protein with the same molecular weight as the dimeric form of the receptor. Measurements of inositol phosphate production showed a rapid functional desensitization of about 90% after agonist treatment, whereas treatment with PMA caused only a 30% loss in the same time. The extent of receptor phosphorylation following the different treatments paralleled the desensitization of the receptor. These results strongly suggest that phosphorylation of the dimeric form of mGluR1c, as a functionally active form, may play a role in its rapid desensitization.