The internalization of nerve growth factor by high-affinity receptors on pheochromocytoma PC12 cells.

The internalization and subsequent fate of the two populations of nerve growth factor (NGF) receptors on pheochromocytoma PC12 cells were explored either by identifying the relative amounts and sizes of the receptors, after incubation of cells with [125I]NGF, by cross-linking with a photoreactive heterobifunctional reagent or by following the topological distribution of the cross-linked receptors with time. The ratio of the slow, high-affinity to the fast, low-affinity NGF receptor decreased over a 5-h incubation with [125I]NGF in a process which did not involve proteolytic conversion of the slow to the fast receptor. During this period the cross-linked slow receptor moved from a trypsin-labile to a trypsin-stable site suggestive of internalization. In contrast, the cross-linked fast NGF receptor remained trypsin sensitive for at least 2 h of incubation, indicative of a constant cell surface localization. The internalized [125I]NGF in the cross-linked slow NGF receptor was not degraded, indicating that cross-linking, by preventing the acid pH-induced dissociation of the NGF-receptor complex in the endosomes, blocks normal sorting of [125I]NGF to the lysosomes. The cross-linked receptor was not recycled to the cell surface. If this reflects the properties of the unmodified receptor then another process, possibly receptor conversion, is required to replenish slow NGF receptors in the cell surface.     

Characterization of heparin-binding growth-associated factor receptor on NIH 3T3 cells.

Scatchard plot analysis of the binding of 125I-labeled heparin binding cell growth-associated factor (125I-HBGAF) to NIH 3T3 cells revealed a single class of high affinity receptors (-5000/cell) with kd of -0.6 nM. 125I-HBGAF was covalently cross-linked to the cell surface receptor on NIH 3T3 cells with disuccinimidyl suberate (DSS). Two 125I-HBGAF-cross-linked complexes of 170 kDa and 142 kDa were observed on SDS-polyacrylamide gel electrophoresis under reducing and nonreducing conditions. The 125I-HBGAF-cross-linked complex formation was completely abolished in the presence of greater than or equal to 100-fold excess of unlabeled HBGAF but not PDGF, EGF, aFGF, bFGF, or insulin. 125I-HBGAF appeared to undergo rapid internalization and relatively slow degradation following binding to the HBGAF receptor on NIH 3T3 cells. These results suggest that NIH 3T3 cells express a high affinity HBGAF receptor which shows two different estimated molecular masses of -155 kDa and -127 kDa. This high affinity HBGAF receptor was also found to express in other cell types.     

Kinetic analyses of the binding of leukemia inhibitory factor to receptor on cells and membranes and in detergent solution.

The equilibrium and kinetic properties of leukemia inhibitory factor (LIF) binding to a range of cell types have been compared. When binding was examined at 4 degrees C, the majority of cells were found to express a single class of high affinity LIF receptor (KD = 20-100 pM; ka = 2-8 x 10(8) min-1 M-1; kd = 0.0004-0.0011 min-1). In contrast, certain activated macrophage populations expressed apparently independent classes of high and low affinity LIF receptor. The low affinity receptors differed from the high affinity receptors in terms of the dissociation rate of the receptor-ligand complex (KD = 1-2 nM; ka = 3-7 x 10(8) min-1 M-1; kd = 0.30-0.67 min-1). At 37 degrees C, the interaction of LIF with its high affinity receptor was more complicated, since occupied LIF receptors were internalized more rapidly than unoccupied receptors, internalized LIF was hydrolyzed and released from the cell, and new receptors were synthesized and expressed on the cell surface. Interestingly, when membranes were prepared from cells that expressed only high affinity receptors, both high and low affinity receptors were detected, while after detergent solubilization of membranes only low affinity receptors were apparent. These results are discussed in terms of a structural model for the LIF receptor in which interaction of a low affinity binding subunit and a second nonbinding subunit is required for the generation of the high affinity receptor.     

Primary cultures of mouse spinal cord express the neonatal isoform of the inhibitory glycine receptor

Expression of the inhibitory glycine receptor complex was investigated in primary cultures of fetal mouse spinal cord using sensitive immunomethods. In these cells, glycine receptor is predominantly of the neonatal isoform characterized by a low affinity for the antagonist strychnine. It contains a ligand binding subunit that differs from that of the adult receptor in antigenic epitopes and apparent molecular weight. Whereas in vivo the neonatal receptor isoform is completely replaced by the adult isoform within 3 weeks after birth, this exchange of subtypes is not seen in culture. The increased expression of the cytoplasmic glycine receptor-associated polypeptide of 93 kd occurring after birth is also seen under culture conditions. Purification of glycine receptor from cultures yielded polypeptides of 49 kd and 93 kd, suggesting that the membrane-spanning core of the neonatal receptor may be a homooligomer composed of 49 kd subunits. About half of the 49 kd subunit is cleaved by trypsinization of the cultures, indicating a predominant cell surface localization of the receptor. Pulse-labeling experiments revealed the 49 kd subunit to be a metabolically stable glycoprotein (half-life approximately 2 days). After its synthesis, a transition time of 30-45 min is required for acquisition of a strychnine binding conformation.     

Subunit structure of the glucocorticoid receptor and activation to the DNA-binding state

Glucocorticoid receptors of S49.1 mouse lymphoma cells were analyzed under a variety of conditions. The complexes with an agonist or a steroidal antagonist can be formed in cytosolic extracts, they are of high molecular weight, Mr approximately 330,000 and have a Stokes radius of 82 A. Cross-linking by several agents stabilized this structure against subunit dissociation which produces the activated receptor form of 60 A and DNA-binding ability. Careful analysis of intermediate cross-linked forms lead to the conclusion that the large receptor structure is a hetero-tetramer consisting of one hormone-bearing polypeptide of Mr approximately 94,000, two 90 kDa subunits and a protein component of Mr approximately 50,000. The 90 kDa subunits are the heat shock protein hsp90. The high molecular weight receptor form also exists in intact cells as revealed again by cross-linking. The cytosolic complex with the antagonist can become activated to the DNA-binding form upon warming but simultaneously looses the ligand. Ligand rebinding does not occur subsequent to receptor dissociation. Upon incubation of intact cells at 37 degrees C with agonist or antagonist the respective receptor-ligand complexes are formed. The agonist complex is immediately activated, however, the antagonist complex remains stable in the undissociated state. This explains the biological effect of the antagonist.     

Differential modulation of two interferon-α binding proteins on a human lymphoblastoid cell line

The interaction between human interferon (IFN)-α or IFN-β with its receptor was originally described as the binding to a single class of high-affinity receptors. However, more recently, biphasic Scatchard plots as well as multiple IFN-α receptor cross-linked complexes have been reported. In this study using the Daudi B lymphoblastoid cell line, two primary IFN-α receptor cross-linked complexes with apparent M_r of 115 and 135 kilodaltons (kDa) were obtained. Both complexes were observed under a variety of cross-linking conditions, including the addition of a mixture of protease inhibitors throughout the binding reaction and solubilization of the cells. These two complexes appear to be caused by the binding and cross-linking of ¹²⁵I-rIFN-αA to two separate proteins because we also observed two IFN-α binding proteins using a ligand-blotting technique. At low concentrations of ¹²⁵I-rIFN-αA, it was found that the intensity of the signal in the 135-kDa cross-linked complex was greater than that of the 115-kDa complex. Addition of increasing concentrations of unlabeled rIFN-αA to a 4°C binding reaction reversed the ratio in intensities of the two complexes. Moreover, after pretreatment of the cells at 37°C with low concentrations of unlabeled rIFN-αA, there was preferential down-regulation of both the 135-kDa complex and the higher affinity binding component of the biphasic Scatchard plot. These results suggest that the 135-kDa complex represents the binding of ¹²⁵I-rIFN-αA to a protein having higher affinity for IFN than the protein that gives rise to the 115-kDa complex. These two proteins also appear to have different half lives in the plasma membrane in the absence of IFN because treatment with cycloheximide also caused a preferential decrease in the subsequent formation of the 135-kDa complex.     

Nerve growth factor binds to the 140 kd trk proto-oncogene product and stimulates its association with the src homology domain of phospholipase C gamma 1.

The cellular actions of nerve growth factor (NGF) involve regulation of protein phosphorylation. In PC-12 pheochromocytoma cells, exposure of [125I]NGF followed by crosslinking indicates that the ligand binds to two discreet receptors, the previously described 75 kd protein, as well as the trk proto-oncogene product pp140c-trk. Competition experiments reveal that of the two, pp 140c-trk binds to NGF with higher affinity. Following exposure to NGF, pp140c-trk undergoes a rapid autophosphorylation on tyrosine residues, and concomitantly phosphorylates and associates with phospholipase C gamma 1 (PLC gamma 1), through interaction with its src homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent homology domains. The binding of NGF to pp140c-trk with high affinity, the NGF-dependent activation of its tyrosine kinase activity and the specific association with the effector molecule, PLC gamma 1, suggests that this is the biologically relevant signaling receptor for NGF.     

MPF from starfish oocytes at first meiotic metaphase is a heterodimer containing one molecule of cdc2 and one molecule of cyclin B.

We have purified to near homogeneity the M-phase-specific protein kinase from starfish oocytes at first meiotic metaphase, using an improved procedure based on affinity chromatography on the immobilized yeast protein suc1. As already reported, this is identical to MPF, the cytoplasmic factor that controls entry of eukaryotic cells into M-phase. MPF is a complex formed by the stoichiometric association of a 34-kd polypeptide previously identified as cdc2 with a polypeptide that migrates with the same mobility as starfish cyclin in SDS-PAGE (apparent mol. wt 47 kd). A cDNA clone encoding starfish cyclin B has been isolated and its sequence determined. It contains a single open reading frame encoding a predicted 43 729-dalton protein. Partial microsequencing of the 47-kd polypeptide component of MPF allowed its identification as the starfish cyclin. Since the apparent mol. wt of native starfish MPF was found to be less than 100 kd, it is a heterodimer comprising one molecule of cdc2 and one molecule of cyclin B.     

Molecular investigations on the high-affinity nerve growth factor receptor.

Nerve growth factor (NGF) receptors have been investigated by means of affinity labeling with 125I-NGF and chemical cross-linking. Two distinct NGF-receptor complexes are detected on PC12 cells; these correspond to 100 kd and 158 kd for the low-affinity (LNGFR) and the high-affinity (HNGFR) receptors, respectively. Interestingly, three different antibodies directed against distinct epitopes on the LNGFR immunoprecipitate the low-but not the high-affinity NGF-receptor complex. Although the identities of the signaling molecules in the HNGFR are unknown, antibodies to the src, ras, raf-1, and yes products fail to immunoprecipitate either receptor complex, suggesting that these molecules are not a part of, or tightly coupled to, either receptor type. Phosphotyrosine residues are found exclusively on the HNGFR complex, suggesting that tyrosine phosphorylation may be one of the initiating events in the NGF-induced signal transduction cascade.     

The trk proto-oncogene encodes a receptor for nerve growth factor.

Two classes of receptors with distinct affinities for nerve growth factor (NGF) have been identified. The low affinity receptor (Kd approximately 10(-9) to 10(-8) M) is a cysteine-rich glycoprotein encoded by the previously characterized LNGFR gene. The structural nature of the high affinity receptor (Kd approximately 10(-11) to 10(-10) M) has yet to be established. In this study we show that the product of the human trk proto-oncogene (gp140trk) binds NGF with high affinity. Moreover, NGF could be chemically cross-linked to the endogenous gp140trk present in rat PC12 pheochromocytoma cells as well as to gp140trk ectopically expressed in mouse fibroblasts and in insect Sf9 cells. High affinity binding of NGF to gp140trk can occur in the absence of low affinity LNGFR receptors, at least in nonneural cells. Addition of NGF to PC12 cells elicits rapid phosphorylation of gp140trk on tyrosine residues and stimulates its tyrosine kinase activity. These results indicate that gp140trk is a functional NGF receptor that mediates at least some of the signal transduction processes initiated by this neurotrophic factor.     

Purification of a pituitary receptor for somatostatin. The utility of biotinylated somatostatin analogs.

A somatostatin (SRIF) receptor and its associated Gi regulatory proteins was purified from GH4C1 rat pituitary cells by: 1) saturation of the membrane-bound receptor with biotinyl-NH-[Leu8,D-Trp22,Tyr25] SRIF28 (bio-S28); 2) solubilization of receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine (D.L); 3) adsorption of solubilized receptor-ligand complex to immobilized streptavidin; and 4) elution of receptor and G-protein by GTP. The receptor, a glycoprotein with an average M(r) of 85,000, was then purified to substantial homogeneity on immobilized wheat germ agglutinin. The 85-kDa glycoprotein was identified as a SRIF receptor by several criteria. (a) It had the same size as the chemically cross-linked R.[125I]L complex. (b) Yield of the purified protein increased and plateaued in the same range of bio-S28 concentrations where specific high affinity binding reached saturation. (c) It was copurified with appropriate G-protein subunits. The 85-kDa receptor and two other proteins with M(r) values of 35,000 and 40,000, the sizes of G beta and G alpha, did not appear in eluates from control streptavidin columns done with SRIF receptors loaded with nonbiotinylated S14. The 40-kDa protein was identified as a Gi alpha by ADP-ribosylation from [32P]NAD catalyzed by pertussis toxin. (d) Both the chemically cross-linked R.[125I]L complex and SRIF receptor purified from [35S]methionine-labeled GH4C1 cells were reduced in size to about 38 kDa by endoglycosidase F. (e) Amino acid sequence from the purified receptor was nearly identical with that of a recently cloned SRIF receptor subtype.     

The Duffy receptor family of Plasmodium knowlesi is located within the micronemes of invasive malaria merozoites

Plasmodium vivax and Plasmodium knowlesi merozoites invade human erythrocytes that express Duffy blood group surface determinants. A soluble parasite protein of 135 kd binds specifically to a human Duffy antigen. Using antisera affinity purified on the 135 kd protein, we cloned a gene that encodes a member of a P. knowlesi family of erythrocyte binding proteins. The gene is a member of a family that includes three homologous genes located on separate chromosomes. Two genes are expressed as major membrane-bound products that give rise to soluble erythrocyte binding proteins: the 135 kd Duffy binding protein and a 138 kd protein that binds only rhesus erythrocytes. These different erythrocyte binding specificities may result from sequence divergence of the homologous genes. The Duffy receptor family is localized in micronemes, an organelle found in all organisms of the phylum Apicomplexa.     

Direct probing of the interaction between the signal sequence of nascent preprolactin and the signal recognition particle by specific cross-linking

We have studied the interaction between the signal sequence of nascent preprolactin and the signal recognition particle (SRP) during the initial events in protein translocation across the endoplasmic reticulum membrane. A new method of affinity labeling was used, whereby lysine residues, carrying the photoreactive group 4-(3-trifluoromethyldiazirino) benzoic acid in their side chains, are incorporated into a protein by means of modified lysyl-tRNA, and cross-linking to the interacting component is induced by irradiation. SRP interacts through its Mr 54,000 polypeptide component with the signal sequences of nascent preprolactin chains containing about 70 residues, and with decreasing affinity with longer chains as well; it causes inhibition of elongation. Binding of SRP is reversible and requires the nascent chain to be bound to a functional ribosome. SRP cross-linked to the signal sequence still inhibits elongation but does not prevent it completely. We conclude that SRP does not block the exit site of the polypeptide chain on the ribosome. The SRP receptor of the endoplasmic reticulum membrane displaces the signal sequence from SRP and, even if SRP is cross-linked, releases elongation arrest.     

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.     

Characterization of the cell surface receptors for human B cell growth factor of 12,000 molecular weight

Quiescent normal human B cells have been shown to require an activation step before proliferating in response to B cell growth factor (BCGF) of 12,000 m.w. (12 kd). One effect of cell activation has been the putative acquisition of specific cell surface growth factor receptors. In this report, the existence of such receptors has been confirmed by using purified radioiodinated BCGF-12 kd. BCGF-12 kd receptors on activated B cells have been shown to be distinct form those interacting with IL 2. Scatchard analysis revealed both high affinity receptor sites with an apparent Kd of 28.6 pM and low affinity receptor sites with Kd of 1.2 nM on freshly prepared, anti-IgM activated peripheral blood B cells. Human B cells grown in culture for extended periods of time in the presence of BCGF-12 kd also displayed high affinity receptor sites (Kd, 41.4 pM) and low affinity receptor sites (Kd, 0.9 nM). The action of BCGF-12 kd therefore appears to be mediated by binding to its lineage-specific receptors on the cell surface.     

Activation of phosphatidylinositol-3 kinase by nerve growth factor involves indirect coupling of the trk proto-oncogene with src homology 2 domains.

Growth factor receptor tyrosine kinases can form stable associations with intracellular proteins that contain src homology (SH) 2 domains, including the p85 regulatory subunit of phosphatidylinositol (PI)-3 kinase. The activation of this enzyme by growth factors is evaluated in PC12 pheochromocytoma cells and NIH 3T3 fibroblasts expressing the pp140c-trk nerve growth factor (NGF) receptor (3T3-c-trk). NGF causes the rapid stimulation of PI-3 kinase activity detected in anti-phosphotyrosine, but not in anti-trk, immunoprecipitates. This effect coincides with the tyrosine phosphorylation of two proteins, with molecular masses of of 100 kd and 110 kd, that coimmunoprecipitate with p85. Similar phosphorylation patterns are induced when an immobilized fusion protein containing the amino-terminal SH2 domain of p85 is used to precipitate tyrosine-phosphorylated proteins. Thus, although NGF produces the rapid activation of PI-3 kinase through a mechanism that involves tyrosine phosphorylation, there is no evidence for tyrosine phosphorylation of p85, or for its ligand-dependent association with the NGF receptor. Perhaps another phosphoprotein may link the NGF receptor to this enzyme.     

The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor

The transforming growth factor-beta (TGF-beta) receptor type III is a low abundance cell surface component that binds TGF-beta 1 and TGF-beta 2 with high affinity and specificity, and is present in many mammalian and avian cell types. Type III TGF-beta receptors affinity-labeled with 125I-TGF-beta migrate in sodium dodecyl sulfate-polyacrylamide electrophoresis gels as diffuse species of 250-350 kDa. Here we show that type III receptors deglycosylated by the action of trifluoromethanesulfonic acid yield affinity-labeled receptor cores of 110-130 kDa. This marked decrease in molecular weight is also achieved by combined treatment of type III receptors with heparitinase and chondroitinase ABC. Digestion of receptor-linked glycosaminoglycans by treatment of intact cell monolayers with heparitinase and chondroitinase does not prevent TGF-beta binding to the type III receptor core polypeptide and does not release the receptor polypeptide from the membrane. The type III TGF-beta receptor binds tightly to DEAE-Sephacel and coelutes with cellular proteoglycans at a characteristically high salt concentration. Thus, the type III TGF-beta receptor has the properties of a membrane proteoglycan that carries heparan and chondroitin sulfate glycosaminoglycan chains. The binding site for TGF-beta appears to reside in the 100-120-kDa core polypeptide of this receptor. The type III receptor is highly sensitive to cleavage by trypsin. Trypsin action releases the glycosaminoglycan-containing domain of the receptor leaving a 60-kDa membrane-associated domain that contains the cross-linked ligand. A model for the domain structure of the TGF-beta receptor type III is proposed based on these results.     

Nerve growth factor induces increased expression of a laminin-binding integrin in rat pheochromocytoma PC12 cells

Rat pheochromocytoma PC12 cells exposed to nerve growth factor differentiate as sympathetic neurons and extend neurites on laminin and to a much lesser extent on fibronectin. Analysis of laminin fragments indicated that neurite outgrowth occurs mainly on fragment P1, corresponding to the center of the cross, and only poorly on fragment E8, a long arm structure that is active with other neuronal cells. Integrin antibodies prevented adhesion and neurite sprouting of these cells on laminin, fragment P1, and fibronectin. By affinity chromatography we isolated an integrin-type receptor for laminin consisting of two subunits with molecular massess of 180 and 135 kDa. The latter is recognized by an antiserum to integrin beta 1 subunit. The bound laminin receptor could be displaced by EDTA, but not by Arg-Gly-Asp or Tyr-Ile-Gly-Ser-Arg peptides. Affinity chromatography on laminin fragments showed that the 180/135 kDa receptor binds to P1. The expression of the 180-kDa alpha subunit of the laminin receptor at the cell surface was increased 10-fold after NGF treatment. The effect of NGF is specific since the amount of a 150-kDa fibronectin-binding integrin alpha subunit remained unchanged. Moreover, the increased expression of the 180/135 kDa receptor at the cell surface corresponded to a selective increase in cell adhesion to laminin and to fragment P1. The 180/135-kDa complex is thus an integrin-type receptor for laminin whose expression and binding specificity correlates with the capacity of NGF-induced PC12 cells to extend neurites on laminin.     

Purification and characterization of the D2 cell adhesion protein: Analysis of the postnatally regulated polymorphic forms and their cellular distribution

The developmentally regulated, D2 cell adhesion protein has been purified from 10–12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5–1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (>200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.     

Protein components of the nonactivated glucocorticoid receptor.

The nonactivated glucocorticoid receptor (Mr approximately 350,000) of WEHI-7 mouse lymphoma cells was investigated with respect to the stoichiometry of protein subunits. Cross-linking patterns obtained by affinity labeling and denaturing gel electrophoresis revealed a heterotetramer consisting of one receptor polypeptide in association with two 90- and one approximately 50-kDa subunits. The receptor stabilized by molybdate, disulfide bond formation, or chemical cross-linking was purified roughly 6000-fold by immunoaffinity chromatography and analyzed by gel electrophoresis and immunoblotting. The 90-kDa component was consistently detected in a 2:1 ratio with respect to the receptor polypeptide and was identified as the 90-kDa heat shock protein, hsp90. A 70-kDa heat shock protein was found in both stabilized and nonstabilized receptors and bound to the immunomatrix independent of receptor. The additional receptor subunit was unequivocally identified as the 59-kDa protein previously described (Tai, P.-K. K., Maeda, Y., Nakao, K., Wakim, N. G., Duhring, J. L., and Faber, L. E. (1986) Biochemistry 25, 5269-5275). This component was found only in complexes cross-linked via amino groups. It was removed from the molybdate-stabilized receptor under our purification conditions, thus leaving behind a trimer composed of the receptor polypeptide and two molecules of hsp90. In the absence of hormone, the receptor had the same subunit composition as in its presence.