Monoclonal Antireceptor Research Articles

Anti-human FSH receptor monoclonal antibodies: immunochemical and immunocytochemical characterization of the receptor.

The extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin. It was tagged with a poly-His tract which was used for its purification. Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies. The latter fell into two categories: some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties. None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase). Immunoaffinity chromatography allowed us to purify the native receptor in a single step either from a permanently transfected L cell line (75% recovery) or from human ovaries (33% recovery). Immunoblotting of the receptor in human ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestion and pulse-chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates. Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells. We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described. Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity approximately 1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein. All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells.

Production and characterization of anti-human interferon gamma receptor antibody fragments that inhibit cytokine binding to the receptor.

Three single-chain antibody fragments that recognize the extracellular human interferon gamma receptor alpha-chain (IFN gamma R), and inhibit the binding of human IFN gamma, have been produced in Escherichia coli. These fragments are derived from murine anti-receptor monoclonal antibodies, and comprise the variable heavy (VH) domain linked to the variable light (VL) chain through a 15 amino acid linker [(GGGGS)3]. Using surface plasmon resonance technology (BIAcore), the soluble proteins were shown to retain a high affinity for recombinant IFN gamma R, and by radioimmunoassay to possess a high inhibitory activity towards IFN gamma-binding to human Raji cells. The antibody fragments most likely recognize epitopes that overlap the cytokine binding site on the receptor surface. Attempts to dissect further the antibodies to isolated VH- and VL-chains and to synthetic linear and cyclic peptides derived from the individual complementarity determining regions failed to afford fragments with significant IFN gamma R binding affinity. Nevertheless, these native-like variable region fragments and petidomimetics derived from them are of interest in the design of novel IFN gamma R antagonists.

Prevention of breast tumour development in vivo by downregulation of the p185neu receptor.

Certain strains of transgenic mice that express the rat neu oncogene (neuT) in mammary epithelial cells develop breast tumours at an average of 44 weeks of age. In this study, intraperitoneal injection of a monoclonal anti-receptor antibody specific for the rat neuT oncogene product dramatically affected tumour development in these transgenic mice in a dose-dependent manner. A significant proportion (50%) of mice, when injected with anti-receptor antibodies, did not develop tumours even after 90 weeks of age. The phosphotyrosine levels of the membrane fraction of breast tissues in the anti-receptor antibody-treated mice were almost completely abolished when a higher dose of antibodies was used. This study demonstrates, for the first time, that immunologic manipulation of an oncogene product can effectively prevent the development of tumours in a rodent transgenic model.

Postsynaptic and extrasynaptic localization of ?-opioid receptor in selected brain areas of young rat and chick using an anti-receptor monoclonal antibody

kappa-opioid receptors were visualized by light and electron microscopical immunohistochemistry in young rat and chick brains, using a monoclonal antibody KA8 (IgG1, kappa) raised against a kappa-opioid receptor preparation from frog brain, which recognizes selectively the kappa-type receptor with preference for the kappa-2 subtype. The most pronounced kappa-opioid receptor-like immunoreactivity was observed in the hypothalamic nuclei of the rat brain and in the chick optic tectum, in regions where the functional significance of kappa-opioid receptors is well documented. Both neurons and glia were stained, the former on both somata and dendrites. At the ultrastructural level, the receptor-like immunoreactivity was similar in both species. Immunoprecipitate decorated the inner surface of the plasma membrane of glial cells, neuronal somata and dendrites, in a discontinuous arrangement. In the cytoplasm, labelling was associated with ribosomes, polyribosomes and rough endoplasmic reticulum membranes but not with Golgi cisternae. In the neuropil, the immunoprecipitate was observed along the dendritic microtubules and was also associated with postsynaptic sites. Nuclei and axons were devoid of label and immunoreactivity was never visible presynaptically. Our findings indicate that the antibody used in the present study marks various forms of the kappa-opioid receptor protein including those synthesised in ribosomes, transported along dendritic microtubules and incorporated into postsynaptic and non-synaptic membranes. The antibody also recognizes glial opioid receptors. The observed subcellular distribution appears to be conserved in phylogenetically distant species.

Response of rat immature enterocytes to insulin: Regulation by receptor binding and endoluminal polyamine uptake

The mechanism(s) by which insulin stimulates enzyme expression in rat immature enterocytes are unknown. The purpose of this study was to investigate these mechanism(s). The effects of insulin or an antireceptor monoclonal antibody (RPN 538) were assessed on microvillous enzyme activities and the endoluminal uptake of [14C]spermine. Changes in de novo synthesis of polyamines were measured by mucosal ornithine decarboxylase activity. In sucklings (day 14), administration of insulin failed to induce intestinal ornithine decarboxylase activity, whereas in older rats (day 18 and 20), ornithine decarboxylase was enhanced by 2-2.5-fold. Immature enterocytes from sucklings, pretreated with alpha-difluoromethylornithine, remained sensitive to insulin and expressed enzyme activities at levels equal to controls. In response to insulin, the uptake of [14C]spermine and the mucosal concentrations of spermine and spermidine were increased by 30%, 13%, and 39%, respectively. Administration of RPN 538 had no effect on [14C]-spermine uptake, but it prevented the effects of endogenous insulin on enzyme expression. The enzymatic response of immature enterocytes to insulin is mediated by binding of the hormone to its receptor and is transduced into the cell without de novo synthesis of polyamines. The regulation by insulin of the endoluminal uptake of spermine could be critical for intestinal maturation.

Phosphorylation of immunopurified rat liver glucocorticoid receptor by the catalytic subunit of cAMP-dependent protein kinase.

We have examined phosphorylation of the rat liver glucocorticoid receptor (GR) and GR-associated protein kinase (PK) activity in the immunopurified receptor preparations. Affinity labeling of hepatic cytosol with [3H]dexamethasone 21-mesylate showed a covalent association of the steroid with a 94 kDa protein. GR was immunopurified with antireceptor monoclonal antibody BuGR2 (Gametchu & Harrison, Endocrinology 114: 274-279, 1984) to near homogeneity. A 23 degrees C incubation of the immunoprecipitated protein A-Sepharose adsorbed GR with [gamma-32P]ATP,Mg2+ and the catalytic subunit of cAMP-dependent PK (cAMP-PK) from bovine heart, led to an incorporation of radioactivity in the 94 kDa protein. Phosphorylation of GR was not evident in the absence of the added kinase. Of the radioinert nucleotides (ATP, GTP, UTP or CTP) tested, only ATP successfully competed with [gamma-32P]ATP demonstrating a nucleotide specific requirement for the phosphorylation of GR. Other divalent cations, such as Mn2+ or Ca2+, could not be substituted for Mg2+ during the phosphorylation reaction. Phosphorylation of GR was sensitive to the presence of the protein kinase inhibitor, H-8, an isoquinoline sulfonamide derivative. In addition, the incorporation of radioactivity into GR was both time- and temperature-dependent. The phosphorylation of GR by cAMP-PK was independent of the presence of hsp-90 and transformation state of the receptor. The results of this study demonstrate that GR is an effective substrate for action of cAMP-PK and that the immunopurified protein A-Sepharose adsorbed GR lacks intrinsic kinase activity but can be conveniently used for the characterization of the phosphorylation reaction in the presence of an exogenous kinase.

Regulation of epidermal growth factor receptor in NIH3T3/HER14 cells by antireceptor monoclonal antibodies.

The mechanism(s) by which monoclonal antibodies (mAbs) against the epidermal growth factor (EGF) receptor regulate receptor function have been investigated with NIH3T3/HER14 fibroblasts expressing human EGF receptors. Bivalent 225 mAb or monovalent 225 Fab' inhibited transforming growth factor (TGF)-alpha-induced EGF receptor tyrosine phosphorylation and cell proliferation. Culture of HER14 cells with 225 mAb or 225 Fab' did not activate EGF receptor tyrosine kinase when assayed after lysis of cells in SDS sample buffer. However, when cells were cultured with bivalent 225 mAb, but not with monovalent 225 Fab', and were subsequently lysed and further incubated in Triton X-100 lysis buffer containing proteinase and phosphatase inhibitors, receptor phosphorylation was observed. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein after lysis, indicating that it was due to the activation of protein tyrosine kinase. The activity of bivalent 225 mAb was unphysiologic, in contrast with TGF-alpha, in that receptor kinase activation occurred only after cell lysis and with delayed kinetics; serine and threonine phosphorylation did not occur; and down-regulation of EGF receptors was slower. Selective mAb-mediated phosphorylation of tyrosine residues on EGF receptors was sufficient to activate phosphorylation of a SH2 group-bearing substrate, phospholipase C-gamma, indicating that serine/threonine phosphorylation is not required for EGF receptor kinase activity. These studies provide novel insights into the capacity of bivalent mAb to modulate EGF receptor function.

Bacterial Synthesis of Truncated Forms of the Human Vitamin D Receptor and Characterization of Anti-receptor Monoclonal Antibodies

We biosynthesized full-length (amino acids 1-427) and truncated human 1,25-dihydroxyvitamin D 3 receptor proteins that encompassed only the putative DNA binding domain (amino acids 1-112)or the DNA binding domain and parts of the sterol binding domain (amino acids 1-193 and 1-328)in a bacterial expression system. We also prepared monoclonal antibodies against the full-length vitamin D receptor. The binding properties of the monoclonal antibodies were characterized by their ability to bind to full-length and truncated vitamin D receptor protein constructs. Seven of twelve monoclonal antibodies recognized the full-length receptor protein. These antibodies bound to truncated hVDR proteins with decreasing affinities as successive truncations were made from the carboxy-terminal end of the receptor protein. The five remaining monoclonal antibodies recognized the full-length and truncated receptor proteins with equally low affinities. Truncated forms of the vitamin D receptor and region-specific antibodies will be useful in assessing the properties of the receptor.

Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor.

Mouse hepatitis virus (MHV) strain A59 uses as cellular receptors members of the carcinoembryonic antigen family in the immunoglobulin superfamily. Recombinant receptor proteins with deletions of whole or partial immunoglobulin domains were used to identify the regions of receptor glycoprotein recognized by virus and by antireceptor monoclonal antibody CC1, which blocks infection of murine cells. Monoclonal antibody CC1 and MHV-A59 virions bound only to recombinant proteins containing the entire first domain of MHV receptor. To determine which of the proteins could serve as functional virus receptors, receptor-negative hamster cells were transfected with recombinant deletion clones and then challenged with MHV-A59 virions. Receptor activity required the entire N-terminal domain with either the second or the fourth domain and the transmembrane and cytoplasmic domains. Recombinant proteins lacking the first domain or its C-terminal portion did not serve as viral receptors. Thus, like other virus receptors in the immunoglobulin superfamily, including CD4, poliovirus receptor, and intercellular adhesion molecule 1, the N-terminal domain of MHV receptor is recognized by the virus and the blocking monoclonal antibody.

Ryanodine Receptor Expression in Embryonic Avian Cardiac Muscle

The cardiac ryanodine receptor serves aa a sarcoplasmic reticulum calcium release channel and contributes to the rise in cytosolic calcium necessary for contraction of the heart. We have investigated the presence and oligomeric form of the ryanodine receptor during embryonic development of the avian heart. A single isoform of the ryanodine receptor was identified both in adult and in embryonic cardiac tissue from Days 4 to 20 of development, using anti-receptor monoclonal antibodies in conjunction with [ 3H]ryanodine binding. The results of sucrose density gradient sedimentation analysis and [ 3H]ryanodine binding indicated that the cardiac ryanodine receptor is present in a tetrameric form in both the adult and embryos at Day 6 of development. The observation of specific [ 3H]ryanodine binding in hearts from Days 4 and 5 of embryonic development also indicates the presence of a tetrameric receptor protein. Although the heart begins to beat at approximately 33-38 hr (Day 1.5) of embryonic development, we were unable to detect the cardiac ryanodine receptor, using biochemical or immunological techniques, prior to Embryonic Day 4. Ryanodine was found to alter the chronotropic state of intact hearts as early as Hamburger and Hamilton stages 15-19 (Embryonic Day 3) suggesting that receptor protein may be present in a limited subset of cells involved in pacemaker activity in the very early embryo. After Embryonic Day 4, ryanodine also exerted a negative inotropic effect on embryonic hearts. Our results suggest that a single isoform of the ryanodine receptor is present from Day 4 to Day 20 of embryonic development in avian cardiac muscle and that the ryanodine receptor assumes a tetrameric structure capable of forming a functional calcium release channel that participates in excitation-contraction coupling as early as Embryonic Day 4. In addition, a ryanodine receptor-related function may serve as a determinant of chronotropic effects in the very early embryonic heart.

Octreotide (SMS 201–995) inhibits the growth of colon peritoneal carcinomatosis in BDIX rats

In order to assess the effect of octreotide, a somatostatin analogue, on the growth of colon peritoneal carcinomatosis, 20 BDIX rats were injected i.p. with 1·10 6 colon cancer cells (DHD/K12 tumor cell line) and received octreotide, 65 μg/kg s.c. every 12 h ( n=10) or saline ( n=10) for 42 days, starting 3 days after tumor cell injection. Animals were killed at the end of the treatment. The mean volume of ascites was lower in the octreotide group ( 33.7±7.6 ml ), than in the control group ( 67.5±16.3 ml ; P<0.05). The extent of peritoneal carcinomatosis (in five classes according to a previously published classification) was lower in the octreotide group ( P<0.05). Cell proliferation, using the BrdU technique, was markedly inhibited by octreotide (labeling index of tumor cells: 17.0±0.6% vs. 26.3±2.2% in controls, P<0.001). No significant decrease in labeling index was observed in normal colonic mucosa. Two subtypes of somatostatin receptors were found in all tumors, using the 30F3 monoclonal antireceptor antibody. K D and B max values were not significantly different in the octreotide and control groups: high affinity, low capacity receptors ( K D =1.4·10 −10 M and 0.7·10 −10 M , respectively; B max =3.8 and 2.9 pmol/mg protein, respectively); low affinity, high capacity receptors ( K D =1±0.2·10 −9 M , respectively; B max =27.8±0.1 and 22.8±0.05 pmol/mg protein, respectively). Octreotide inhibits the growth of colon peritoneal carcinomatosis in vivo in BDIX rats through an inhibition of DNA synthesis; this effect is at least partly mediated by somatostatin receptors present on tumor cells.

Soluble c-kit proteins and antireceptor monoclonal antibodies confine the binding site of the stem cell factor.

The binding of the stem cell factor (SCF) to the c-kit-encoded receptor tyrosine kinase stimulates a variety of biochemical responses that culminate in cellular proliferation, migration, or survival. The extracellular domain of p145kit consists of five immunoglobulin-like domains. To confine the ligand binding site to this portion of the receptor we generated a panel of murine monoclonal antibodies (mAbs) to the Kit protein and identified two mAbs that efficiently displaced receptor-bound SCF and also inhibited proliferation of SCF-dependent human megakaryocytes. To map the epitopes of these mAbs we constructed and expressed soluble portions of the extracellular domain of Kit, which included either the two amino-terminal Ig-like domains (denoted Kit 1-2), three Ig-like domains (Kit 1-2-3), or the entire extracellular portion (Kit-X). All three recombinant proteins were recognized by the ligand inhibitory mAbs, suggesting that the SCF binding site resides in the amino-terminal half of the ecto-domain. Consistent with this conclusion, all of the soluble proteins inhibited SCF binding to Kit-expressing cells, and they also underwent specific covalent cross-linking to the radiolabeled ligand. However, whereas Kit 1-2-3 and Kit-X displayed comparable ligand affinities, deletion of the third Ig-like domain, in Kit 1-2, involved significant reduction in SCF binding. Hence, the binding site of SCF probably includes Ig-like domains 1 and 2, but structural determinants distal to this portion may also participate in ligand recognition.

Immunohistochemical determination of endometrial progesterone receptor (PR) content after intrauterine infusion of danazol in rabbits

Danazol, an isoxazole derivative of 17 alpha-ethinyl-testosterone, has various effects on the female reproductive system. To examine its anti-estrogenic effects through the receptor system and clarify its direct effect on the endometrium in vivo, we applied danazol jelly into the rabbit uterine cavity. Recent studies performed in our laboratory using the ligand-binding assay have shown that danazol administered directly into the uterine cavity is absorbed by the endometrial tissue and exerts its anti-estrogenic effects possibly through progesterone receptors (PR) in the cells. In the present study we examined the variations in endometrial PR content during 7 days after the intrauterine administration of danazol into the rabbit uterine cavity. Immature female white rabbits were used. They were given daily subcutaneous injections of estradiol-17 beta (E2) for 3 days after which their abdomens were opened, and danazol gel was infused into the lumen of the right horn and HPC-jelly was infused into the lumen of the left horn, as the control. Thereafter and until they were killed, the same daily dose of E2 was injected. They were killed 6h, 24h, 3 days, 5 days or 7 days after intrauterine infusion of danazol and PRs were studied immunohistochemically using a specific antireceptor monoclonal antibody (PR-AT 4.14). Six hours after danazol infusion, the PR staining in the glandular epithelium and part of the stroma surrounding the epithelium had declined. On day 3, PR staining declined to its lowest level in the glandular epithelium and stroma. On Day 5, PR staining had been restored in the glandular epithelium but stayed low in the stroma.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of the full-length rabbit prolactin receptor and its specific domains in baculovirus infected insect cells

The prolactin receptor is a membrane protein mainly involved in the development of the mammary gland and in lactation in mammals. We used specific cDNA constructs and the insect/baculovirus expression system and produced independently and in large amounts several recombinant forms of the rabbit mammary gland prolactin receptor: the full-length receptor (L1, L2), a truncated membranes form (S), a secretable form of the extracellular domain (E) and two forms of the intracellular domain (I1, I2). Of these forms, the L1 and L2 are associated with the membrane fraction, the E is predominantly secreted into the medium and the I1 and I2 are expressed as soluble proteins and surprisingly, a great portion accumulates in the culture medium. The molecular mass (94 kDa) of the expressed full-length receptor corresponds to the translation product of the entire cDNA coding region. The receptor biochemically identified in the rabbit mammary gland is however much shorter. Thus, in the mammary gland, the receptor presumably undergoes post-translational modifications. The receptor forms L1, L2 and S bind prolactin with specificity and affinity similar to those reported for the native receptor. They also interact with two monoclonal antibodies, M 110 and A 917, specific for the native conformation of the hormone-binding site. The I1 and I2 forms do not bind prolactin, whereas the E form does. Thus, the hormone binding site is located in the extracellular domain which can function autonomously as a PRL-binding soluble protein. However, the E form binds prolactin with a higher affinity than the native receptor and it does not bind one of the two antireceptor monoclonal antibodies, known to be hormone binding-site specific. Thus, the conformation of the native receptor and that of the E form differ.

Human anti-acetylcholine receptor antibodies use variable gene segments analogous to those used in autoantibodies of various specificities

The production of autoantibodies to the nicotinic acetylcholine receptor are responsible for many of the neurological symptoms observed in myasthenia gravis. An understanding of the structural organization of the anti-receptor antibodies may help to define the role of these antibodies in the pathogenesis of this disease. The nucleotide sequences of the heavy and light chains of three human monoclonal anti-receptor antibodies isolated from peripheral blood lymphocytes from two patients with myasthenia gravis were analyzed. In addition, the structure of an anti-idiotypic antibody was studied. The V H and V L gene segments used in the anti-receptor antibodies appear to be derived from the same repertoire as gene segments that have been found in other autoantibodies isolated from patients with various autoimmune diseases. The IgM anti-receptor antibodies are direct copies of germline gene segments, while the structures of the IgG anti-receptor antibody and the anti-idiotypic antibody appear to be mutated suggesting that they have undergone antigenic selection.

Screening for hybridomas producing antibodies to steroid hormone receptors: interference from phenol red in the hybridoma culture supernates.

The culture supernates from a variety of mouse hybridomas which were known to secrete antibodies to antigens unrelated to steroid hormone receptors were examined for routine use as experimental controls for monoclonal anti-oestrogen receptor (ER) antibodies. During this process, we made an observation which is important for all investigations on monoclonal anti-receptor antibodies. Our results indicated that the phenol red dye present in these hybridoma culture fluids by binding to the immunoglobulins (Ig) secreted by the hybridoma cells, may impart to the Ig-dye complex, a capacity to interact with ER/progesterone receptors(PR) which are present in the cytosols or in the sections of ER+ tumors. The Ig-dye may thus mimic anti-receptor antibody activity. Growth media containing phenol red and Ig-free bovine calf serum supplement or culture fluids from a non-Ig secreting hybridoma failed to show similar binding to ER+ tissues. This observation documents the need for Ig-dye complex formation to obtain such ER-Ig interaction. Complete dialysis of phenol red from the Ig positive supernates removed the observed interference of the dye. In this report we describe the nature of this interference and the results obtained before and after the removal of the dye from Ig+ culture fluids. In addition, we suggest some remedial measures that should be considered, while screening and testing the specificity of any hybridoma culture product for anti-receptor antibody activity (particularly, anti-ER or anti-PR antibodies).

Pathways of internalization of the hCG/LH receptor: immunoelectron microscopic studies in Leydig cells and transfected L-cells.

Monoclonal anti-receptor antibodies were used to study the cellular traffic of the hCG/LH receptor by immunoelectron microscopy. The LHR38 antibody was shown to bind to the extracellular domain of the receptor but not to interfere with hormone binding, adenylate cyclase activation or with the rate of internalization of the receptor. Pig Leydig cells and a permanent L-cell line expressing the LH receptor were used for the study. Incubation with LHR38-gold complexes showed the LH receptors to be randomly distributed over the cell surface including the clathrin coated pits. The LH receptors were internalized via a route including coated pits, coated vesicles and multivesicular bodies to lysosomes. This route is different from that observed for beta-adrenergic, muscarinic, and yeast mating factor receptors and considered previously as possibly general for G-protein-coupled receptors. The use of [125I]LHR38 allowed precise measurement of the rate of internalization, showing the existence of a constitutive pathway which was increased 11-fold by hormone administration. Double labeling experiments suggested that the hormone (hCG-Au15nm) and the receptor (labeled with LHR38-Au5nm) have similar routes of endocytosis, both of them being degraded in lysosomes. Studies of the reappearance of LHR38-Au5nm on the surface of the cells and the use of monensin indicated that only a very small proportion of the receptor molecules were recycled to the cell surface. The distribution and the intracellular pathways of LH receptors are very similar in Leydig cells and transfected L-cells. This opens the possibility of using the latter to study, by in vitro mutagenesis, the molecular mechanisms involved in the cellular traffic of LH receptors.

Localization of AMPA receptors in the hippocampus and cerebellum of the rat using an anti-receptor monoclonal antibody

The primary amino acid sequences of the kainate binding proteins from the amphibian and avian central nervous systems are homologous with the functional α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors that have been cloned from rat brain. In this study, we have analysed the anatomical and subcellular distribution of the α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptors in the rat hippocampus and cerebellum, using a monoclonal antibody that was raised against a kainate binding protein purified from frog brain. Immunoblots of rat hippocampus and cerebellum, and membranes from COS cells transfected with rat brain α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor cDNAs (GluRl, GluR2, or GluR3) showed a major immunoreactive band migrating at a relative molecular weight of 107,000. In the cerebellum, an additional immunoreactive protein of approximately 128,000 mol. wt was also seen on immunoblots probed with the antibody. The distribution of this protein is apparently restricted to the cerebellum since the 128,000 mol. wt band was not present in other brain areas examined. The identity of the 128,000 mol. wt cerebellar protein is not known. Immunocytochemical analyses of the hippocampus demonstrated that α-amino-3-hydroxy-5-methyl-isoxazole-4-propionate receptor subunits are present in the cell bodies and dendrites of pyramidal cells. The granule cells were also immunostained. All of the pyramidal cell subfields were heavily labeled. In the pyramidal cell bodies, a high level of immunoreactivity was observed throughout the cytoplasm. In the cerebellum, the Purkinje cell bodies and dendrites also displayed very high levels of immunoreactivity. In addition to the Purkinje neurons, the Bergmann glia and some Golgi neurons were clearly immunostained. Subcellular fractionation and lesioning experiments using the excitotoxin domoic acid indicated that the α-amino-3-hydroxyl-5-methyl-isoxazole-4-propionate receptor subunits were associated with postsynaptic membranes. Direct visualization of the immunoreactivity using electron microscopy confirmed the postsynaptic localization of the staining in the dendritic areas in both the hippocampus and the cerebellum. Thus, unlike the kainate binding proteins, which are found primarily extrasynaptically in the frog and on glial cells in the chicken cerebellum, the GluR1, GluR2, and GluR3 receptor subunits are localized to the postsynaptic membrane in the dendrites of neurons in the rat central nervous system.

The role of insulin receptor kinase domain autophosphorylation in receptor-mediated activities. Analysis with insulin and anti-receptor antibodies.

The role of specific tyrosine autophosphorylation sites in the human insulin receptor kinase domain (Tyr1158, Tyr1162, and Tyr1163) was analyzed using in vitro mutagenesis to replace tyrosine residues individually or in combination. Each of the three single-Phe, the three possible double-Phe a triple-Phe and a triple-Ser mutant receptors, stably expressed in Chinese hamster ovary cells, were compared with the wild-type receptor in their ability to mediate stimulation of receptor kinase activity, glycogen synthesis, and DNA synthesis by insulin or the human-specific anti-receptor monoclonal antibody 83-14. At a concentration of 0.1 nM insulin which produced approximately half-maximal responses with wild-type receptor, DNA synthesis and glycogen synthesis mediated by the three single-Phe mutants ranged from 52 to 88% and from 32 to 79% of the wild-type receptor, respectively. The corresponding figures for the double-Phe mutants averaged 15 and 6%, whereas the triple-mutants were unresponsive in both assays. The level of biological function approximately paralleled the insulin-stimulated tyrosine kinase activity in the intact cell as estimated by tyrosine phosphorylation of the insulin receptor and its endogenous substrate pp 185/IRS-1. Interestingly, all mutants showed a marked decrease in insulin-stimulated receptor internalization. Anti-receptor antibody stimulated receptor kinase activity and mimicked insulin action in these cells. In general, the impairment of the metabolic response was greater and impairment of the growth response was less when antibody was the stimulus. These experiments show that the level and specific sites of autophosphorylation are critical determinants of receptor function. The data are consistent with a requirement for the receptor tyrosine kinase either as an obligatory step or a modulator, in both metabolic and growth responses, and demonstrate the important role of the level of insulin receptor kinase domain autophosphorylation in regulating insulin sensitivity.

Binding of the coronavirus mouse hepatitis virus A59 to its receptor expressed from a recombinant vaccinia virus depends on posttranslational processing of the receptor glycoprotein.

Recently, we showed that a murine member of the carcinoembryonic antigen family of glycoproteins serves as a cellular receptor (MHVR) for the coronavirus mouse hepatitis virus A59 (MHV-A59) (G. S. Dveksler, M. N. Pensiero, C. B. Cardellichio, R. K. Williams, G.-S. Jiang, K. V. Holmes, and C. W. Dieffenbach, J. Virol. 65:6881-6891, 1991; R. K. Williams, G.-S. Jiang, and K. V. Holmes, Proc. Natl. Acad. Sci. USA 88:5533-5536, 1991). To examine the role of posttranscriptional modification of MHVR on virus-receptor interactions, a vaccinia virus-based expression system was employed. Expression from the vaccinia virus recombinant (Vac-MHVR) in BHK-21 cells resulted in high levels of MHVR glycoprotein on the cell surface and made these cells susceptible to MHV-A59 infection. Nonglycosylated core MHVR proteins were made in Vac-MHVR-infected BHK-21 cells in the presence of tunicamycin by in vitro translation of MHVR mRNA in a rabbit reticulocyte cell-free system in the absence of microsomal membranes and by expression of an N-terminal deletion clone of MHVR lacking its signal peptide. These three nonglycosylated MHVR proteins were recognized by polyclonal antibody against affinity-purified receptor but did not bind antireceptor monoclonal antibody (MAb) CC1 or MHV-A59 virions. Partial glycosylation of MHVR, either expressed in Vac-MHVR-infected cells treated with monensin or synthesized by in vitro translation with microsomal membranes, restored both the MAb CC1- and the virus-binding activities of the MHVR glycoprotein. Deletion of 26 amino acids at the carboxyl terminus of MHVR resulted in a secreted protein which was able to bind MAb CC1 and MHV-A59. These results suggest that either a carbohydrate moiety is an element of the MHVR-binding site(s) for virus and MAb CC1 or a posttranslational membrane-associated process is required for functional conformation of the receptor glycoprotein.