Photoaffinity labeling of the erythropoietin receptor and its identification in a ligand-free form.

Pure human recombinant erythropoietin (EP) was acylated through a primary amino residue with a cross-linking reagent, N-[[3-[[4-[(p-azido-m-[125I]iodophenyl)azo]benzoyl]amino] propanoyl]oxy]-succinimide (Denny-Jaffe reagent), which is photoreactive and cleavable at the azo residue. The resulting conjugated hormone (DJ-EP) was purified from unmodified EP by reverse-phase high-pressure liquid chromatography and maintained its capacity to bind to receptors for EP on erythroid progenitor cells. The receptor for EP was previously identified as two related proteins of 100 and 85 kDa molecular mass by chemical cross-linking to 125I-EP. Recently, D'Andrea and co-workers [(1989) Cell 57, 277-285] cloned a cDNA that codes for a protein of 55-66 kDa, which is thought to be the EP receptor. In this report, cross-linking to the receptor through the monofunctional DJ-EP labeled the same 140- and 125-kDa molecular mass bands (100- and 85-kDa proteins) cross-linked with 125I-EP and disuccinimidyl suberate. Furthermore, cleavage of the azo bond of the DJ-EP receptor complex by sodium dithionite (80 degrees C, 5 min) demonstrated that proteins of 105 and 90 kDa were labeled in ligand-free form by DJ-EP. This result demonstrates that artifactual cross-linking of multiple proteins or other artifacts of cross-linking do not explain the difference in molecular mass of the EP receptor identified by cross-linking and the receptor identified by expression cloning.     

Structure of the murine erythropoietin receptor complex. Characterization of the erythropoietin cross-linked proteins.

The structure of the murine erythropoietin receptor was studied using antibodies against the intracellular part of the cloned erythropoietin receptor chain. These antibodies precipitated erythropoietin-receptor complexes from Triton X-100-solubilized cells. When the complexes were cross-linked by disuccinimidyl suberate, the 85- and 100-kDa erythropoietin-cross-linked proteins previously described were immunoprecipitated. However, these proteins were not precipitated when the complexes were denatured and reduced before immunoprecipitation. Using 1-ethyl 3-(3-dimethylaminopropyl)carbodiimide, we observed erythropoietin cross-linking with a protein of 66 kDa in addition to the 100- and 85-kDa proteins. Only the 66-kDa erythropoietin-cross-linked protein was immunoprecipitated by anti-receptor antibodies after denaturation and reduction of the complex. Thus, our results suggest that the 85- and 100-kDa proteins previously evidenced by cross-linking are associated with the cloned chain of the receptor to form a multimeric complex but these proteins seem immunologically unrelated to the cloned chain. We observed that reducing the length of molecules able to cross-link amino groups decreased the efficiency of cross-linking with the 100-kDa protein and only the 85-kDa protein was cross-linked with erythropoietin using 1,5-difluoro-2,4-dinitrobenzene. These results suggest that the 85- and 100-kDa proteins occupate slightly different positions relative to the erythropoietin molecule bound to the receptor.     

Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.     

The 260-kDa transforming growth factor (TGF)-beta binding protein in rat glomeruli is a complex comprised of 170- and 85-kDa TGF-beta binding proteins.

In a previous study (MacKay, K., Robbins, A. R., Bruce, M. D., and Danielpour, D. (1990) J. Biol. Chem. 265, 9351-9356) we showed that rat glomeruli contain transforming growth factor (TGF)-beta 1 binding proteins with apparent molecular masses of 260, 170, and 85 kDa (Gl-260, Gl-170, Gl-85) as determined by electrophoresis under nonreducing conditions. We demonstrate here that Gl-260 is a complex of 170- and 85-kDa TGF-beta binding proteins. Under denaturing conditions the integrity of Gl-260 is maintained through the cross-linking of one monomer of the disulfide-linked TGF-beta 1 homodimer to Gl-85 and of the other monomer to the 100-kDa subunit of Gl-170. In addition, some Gl-260 complexes are maintained by direct cross-linking of Gl-85 to the 100-kDa subunit of Gl-170. One-dimensional peptide maps of Gl-85 and the 100-kDa subunit of Gl-170 indicate that they have distinctly different ligand binding domains. In contrast, peptide maps of Gl-85 and the type II receptor of normal rat kidney fibroblasts are similar. The biological responses of isolated glomeruli to TGF-beta appear to parallel those of cultured glomerular cells which are without detectable Gl-170 and Gl-260 binding proteins.     

Structure of the glucocorticoid receptor in intact cells in the absence of hormone.

The nonactivated glucocorticoid receptor (Mr approximately 330,000, Strokes radius = 82 A) contained in cell extracts and complexed with a steroidal ligand was previously investigated by chemical cross-linking. It was identified as a heterotetramer composed of one receptor polypeptide, two molecules of the 90-kDa heat shock protein hsp90, and one 59-kDa protein subunit (Rexin, M., Busch, W., and Gehring, U. (1991) J. Biol. Chem. 266, 24601-24605). We now have used the cross-linking technique to investigate the receptor structure in intact WEHI-7 mouse lymphoma cells at 37 degrees C and under steroid-free conditions. Using immunochemical methods we show that the receptor present in whole cells likewise exists as a high molecular weight structure of Strokes radius 82 A. It has a subunit composition identical to that of the nonactivated receptor-steroid complex in cell extracts. This is the first account of a steroid hormone receptor in its native state as it is contained in target cells under physiological conditions and before a hormonal signal is received.     

A mitogenic fibrinogen receptor that differs from glycoprotein IIb-IIIa. Identification by affinity chromatography and by covalent cross-linking

The mitogenic effect of human fibrinogen on the hemopoietic cell lines Raji and JM is mediated by a specific receptor with biochemical and functional properties different from those of the platelet fibrinogen receptor, the glycoprotein complex IIb-IIIa (Levesque, J.P., Hatzfeld, A., and Hatzfeld, J. (1986) Proc. Natl. Acad. Sci. U.S. A. 83, 6494-6498; Levesque, J.P., Hatzfeld, A., Hudry-Clergeon, G., Wilner, G.D., and Hatzfeld, J. (1987) J. Cell. Physiol. 232, 303-310). This work describes the identification of the mitogenic fibrinogen receptor (MFR) by two independent methods, affinity chromatography and covalent cross-linking. Affinity chromatography of surface-labeled cell extracts on fibrinogen-Sepharose revealed a 94 +/- 2-kDa membrane protein that bound specifically to fibrinogen-Sepharose only on cells that expressed the MFR. Its molecular mass was not modified after reduction. This was confirmed by cross-linking fibrinogen to surface-labeled Raji cells using the cleavable cross-linkers, ethyleneglycobis(succinimidyl succinate) and dithiobis(succinimidyl propionate). Complexes between fibrinogen and iodinated cell membrane proteins were immunoprecipitated by anti-fibrinogen antibodies. The biochemical cleavage of these immunoprecipitated conjugates gave rise to a 92 +/- 3-kDa membrane protein whose molecular mass was not modified after reduction. We conclude that fibrinogen binds specifically to a 92-94-kDa MFR which does not belong to the integrin family.     

Properties of the nerve growth factor receptor. Relationship between receptor structure and affinity

Microsomal membranes from A875 human melanoma cells contain nerve growth factor receptors (NGF-receptors) which appear to belong to a single class with homogeneous binding properties, as determined by Scatchard plots. NGF-receptors in these membrane preparations are also uniformly highly sensitive to tryptic proteolysis, and 125I-NGF bound to NGF-receptor in these membranes is rapidly dissociated in the presence of a high concentration of unlabeled NGF. However, analysis of 125I-NGF dissociation kinetics indicated that two classes of NGF-receptor were present in these membranes. Thus, NGF-receptors can express either high or low affinity trypsin-sensitive states in addition to the high affinity trypsin resistant NGF-receptor state described previously (Buxser, S. E., Kelleher, D. J., Watson, L., Puma, P., and Johnson, G. L. (1983) J. Biol. Chem. 258, 3741-3749). The high affinity trypsin-sensitive and low affinity trypsin-sensitive states correlate with 200- and 90-kDa 125I-NGF X NGF-receptor complexes observed in photoaffinity cross-linking experiments. The absence of differences in peptide maps generated from the two sizes of NGF-receptor proteins together with structural and binding data strongly indicates that the 200-kDa NGF-receptor protein is a complex, probably a dimer, consisting of two 80-kDa NGF-receptor proteins associated with a single beta-NGF dimeric molecule. A model is proposed which relates structural states of NGF-receptors with specific receptor binding properties. The model provides an alternative explanation for binding phenomena previously attributed to negative cooperativity.     

Isoform composition and stoichiometry of the approximately 90-kDa heat shock protein associated with glucocorticoid receptors

We have observed that the approximately 90-kDa non-steroid-binding component of nonactivated glucocorticoid receptors purified from WEHI-7 mouse thymoma cells (which has been identified as the approximately 90-kDa heat shock protein) consistently migrates as a doublet during polyacrylamide gel electrophoresis under denaturing and reducing conditions. It has recently been reported that murine Meth A cells contain a tumor-specific transplantation antigen (TSTA) which is related or identical to the approximately 90-kDa heat shock protein (Ullrich, S.J., Robinson, E.A., Law, L.W., Willingham, M., and Appella, E. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3121-3125). The observation that TSTA and the approximately 90-kDa heat shock protein isolated from these cells exists as two isoforms of similar molecular mass and charge has suggested to us that the doublet we observed is also due to the existence of two isoforms. However, unlike TSTA, which appears to contain the two isoforms in similar relative abundance, nonactivated glucocorticoid-receptor complexes seem to contain predominantly the lower molecular mass isoform. We have therefore conducted this study to determine whether TSTA and the approximately 90-kDa component of glucocorticoid receptors are indeed related, to establish whether the receptor preferentially binds one isoform of the approximately 90-kDa heat shock protein, and to investigate the stoichiometry of the nonactivated receptor complex. By comparing Meth A TSTA and the approximately 90-kDa component of the receptor in their reactions with the AC88 monoclonal antibody (specific for the approximately 90-kDa heat shock protein) and a polyclonal antibody directed against Meth A TSTA, we found that these two proteins are indistinguishable and probably identical. We then used the BuGR1 (directed against the steroid-binding subunit of glucocorticoid receptors) and AC88 monoclonal antibodies to purify, respectively, receptor-associated and free approximately 90-kDa heat shock protein from WEHI-7 cells grown for 48 h with [35S]methionine to metabolically label proteins to steady state. Following analysis of the proteins by polyacrylamide gel electrophoresis under denaturing and reducing conditions, the relative amounts of the two isoforms in each sample were determined from the 35S counts and the known methionine content of each isoform. We found that approximately three-quarters of both the receptor-associated and the free approximately 90-kDa heat shock protein is present as the lower molecular weight isoform, indicating no preferential binding of either isoform in the receptor. The long-term metabolic labeling approach has also enabled us to direc     

Characterization of multiple forms of the Ah receptor: recognition of a dioxin-responsive enhancer involves heteromer formation.

We have employed a combination of gel retardation, protein-DNA cross-linking, and protein-protein cross-linking techniques to further examine the 2,3,7,8-tetrachlorodibenzo-p- dioxin-(TCDD-) dependent changes in the Ah receptor that result in a DNA-binding conformation. Gel retardation analysis of DNA-Sepharose chromatographic fractions of rat hepatic cytosol indicated that TCDD-dependent and sequence-specific DNA binding coeluted with a 200-kDa form of the Ah receptor (peak 2) previously characterized as being multimeric and having high affinity for calf thymus DNA. The TCDD-bound, 100-kDa form of the receptor (peak 1) bound weakly to the DNA recognition motif. These results indicated that the DNA-binding form of the Ah receptor is a multimer. SDS-polyacrylamide gel electrophoresis of peak 2 cross-linked to a bromodeoxyuridine-substituted DNA recognition motif indicated that this form of the receptor present in rat hepatic cytosol is composed of at least two DNA-binding proteins of approximately 100 and 110 kDa. Using the chemical cross-linking agent dimethyl pimelimidate, we further established that the 100-kDa form of the receptor (peak 1) associates with a different protein to generate the receptor form (peak 2) that binds to the dioxin-responsive enhancer. Photoaffinity-labeling studies indicated that only the 100-kDa protein (peak 1), and not the 110-kDa protein, binds ligand. Together, these observations imply that the DNA-binding form of the Ah receptor exists as a heteromer.     

The molybdate-stabilized L-cell glucocorticoid receptor isolated by affinity chromatography or with a monoclonal antibody is associated with a 90-92-kDa nonsteroid-binding phosphoprotein

We have previously reported that molybdate-stabilized cytosol prepared from 32P-labeled L-cells contains two phosphoproteins (a 90-92- and a 98-100-kDa protein) that elute from an affinity resin of deoxycorticosterone-derivatized agarose in a manner consistent with the predicted behavior of the glucocorticoid receptor (Housley, P. R., and Pratt, W. B. (1983) J. Biol. Chem. 258, 4630-4635). In the present work we report that both the 90-92- and 98-100-kDa 32P-labeled proteins are also extracted from molybdate-stabilized cytosol by incubation with a monoclonal antibody and protein A-Sepharose. Only the 98-100-kDa protein is specifically labeled when either L-cell cytosol or L-cell cytosol proteins bound to the affinity resin are labeled with the glucocorticoid binding site-specific affinity ligand [3H]dexamethasone 21-mesylate. The 98-100-kDa protein labeled with [3H]dexamethasone mesylate is adsorbed to protein A-Sepharose in an immune-specific manner after reaction with the monoclonal antibody. Sodium dodecyl sulfate-polyacrylamide gel analysis of the protein A-Sepharose-bound material resulting from incubating the monoclonal antibody with a mixture of 32P-labeled cytosol and [3H]dexamethasone mesylate-labeled cytosol demonstrates identity of the 98-100-kDa [3H]dexamethasone mesylate-labeled band with the 98-100-kDa 32P-labeled band and clear separation from the nonsteroid-binding 90-92-kDa phosphoprotein. The results of immunoblot experiments demonstrate that the 90-92-kDa protein is structurally distinct from the 98-100-kDa steroid-binding protein. As the 90-92-kDa nonsteroid-binding phosphoprotein co-purified with the 98-100-kDa uncleaved form of the glucocorticoid receptor by two independent methods, one of which is based on recognizing a steroid-binding site and the other of which is based on recognizing an antibody binding site, we propose that the 90-92-kDa phosphoprotein is a component of the molybdate-stabilized, untransformed glucocorticoid-receptor complex in L-cell cytosol.     

Disruption of Ha_BtR alters binding of Bacillus thuringiensis delta-endotoxin Cry1Ac to midgut BBMVs of Helicoverpa armigera

Disruption of the Ha_BtR (a cadherin gene) is genetically linked to resistance to Cry1Ac delta-endotoxin of Bacillus thuringiensis in the GYBT strain of Helicoverpa armigera. Brush border membrane vesicles (BBMVs) prepared from midguts of both the Cry1Ac-resistant GYBT strain (homozygous for a deletion knockout of Ha_BtR) and the susceptible GY strain (homozygous for the wild type of Ha_BtR) possessed saturable and specific binding ability to (125)I-Cry1Ac. The binding constant (K(d)) of the GY strain was significantly lower than that of the resistant GYBT strain, whereas their binding site concentrations (B(max)) were similar. When midgut BBMVs were reacted directly with streptavidin conjugated to horseradish peroxidase, the GY strain had very clear 120- and 85-kDa protein bands, which indicated that the 120- and 85-kDa bands are endogenous biotin-containing proteins. However, the GYBT strain almost completely lost these two biotin-containing proteins. Ligand blotting with biotinylated Cry1Ac toxin showed midgut BBMVs of the GY strain contain five protein bands of 210-, 190-, 150-, 120-, and 85-kDa, respectively, while BBMVs of the GYBT strain contain only two protein bands of 150- and 120-kDa. 120-kDa bands may consist of two proteins with coincidentally the same molecular weight (putatively, an APN and a biotin-containing protein). Our results showed that the binding pattern of Cry1Ac to midgut BBMVs of H. armigera was altered quantitatively and qualitatively by knockout of Ha_BtR. There are multiple Cry1Ac-binding proteins in the midgut of susceptible H. armigera, but only the Ha_BtR can be considered as a putative functional receptor of Cry1Ac. Possible involvement of other receptor proteins in the intoxication process in vivo could not be excluded.     

Characterization of the adenovirus E3 protein that down-regulates the epidermal growth factor receptor. Evidence for intermolecular disulfide bonding and plasma membrane localization.

We have characterized the biosynthesis and processing of a 91 amino acid hydrophobic integral membrane protein encoded by human group C adenoviruses which down-regulates the EGF receptor (Carlin, C. R., Tollefson, A. E., Brady, H. A., Hoffman, B. L., and Wold, W. S. M. (1989) Cell 57, 135-144). Previous studies have shown that two immunologically related proteins are produced in vivo, a 13.7-kDa protein encoded by E3 message f and a 11.3-kDa protein derived from 13.7 kDa by proteolysis (Hoffman, B. L., Ullrich, A., Wold, W. S. M., and Carlin, C. R. (1990) Mol. Cell. Biol. 10, 5521-5524; Tollefson, A. E., Krajcsi, P., Yei, S., Carlin, C. R., and Wold, W. S. M. (1990) J. Virol. 64, 794-801). We report here that the 13.7- and 11.3-kDa proteins form intermolecular disulfide bonds cotranslationally at Cys-31 and tend to migrate as high molecular weight aggregates under nonreducing conditions. Both proteins are also present at the cell surface, as evidenced by specific immunoprecipitation from intact monolayers enzymatically labeled with 125I. Moreover, an antiserum specific for a putative extracellular epitope recognizes the same viral proteins as antibodies directed against a C-terminal synthetic 15-mer. The 13.7- and 11.3-kDa proteins are detected at early time points during pulse-chase radiolabeling of infected cells, do not undergo any further changes in molecular weight, and focus at their predicted isoelectric points (7.4 and 7.2, respectively). Identical results are obtained in stable transfectants constitutively expressing only 13.7 and 11.3 kDa, suggesting that biosynthesis and processing is not dependent on other viral proteins. These results have been incorporated into a computer-based model to predict the orientation of 13.7 and 11.3 kDa in the lipid bilayer. This model provides a basis for testing predictions regarding the topology of the viral proteins, as well as putative interactions with heterologous proteins in the microenvironment of the plasma membrane that cause down-regulation of the epidermal growth factor receptor.     

Covalent Cross-Linking of Radiolabeled N-Formylated Hexapeptide to its Specific Receptor on Rat and Human Neutrophils: Evidence for a ligand induced complex formation

Abstract

The structure of the rat and human neutrophil receptor for N-formylated chemotactic peptides was characterized using ¹²⁵I-labeled N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys hexapeptide as a ligand and an affinity cross-linking technique. ¹²⁵I-hexapeptide bound to purified rat peritoneal neutrophils was time, temperature and pH-dependent. The average receptor number per cell was about 67.000 and díssociation constant (K_d) 0.41 nM. Formyl-MLLP, fMLP, fNLP, were 750%, 15%, 8.6% respectively and Boc-MLP, Boc-NLP, and MLP 0.6% as potent as the hexapeptide in inhibiting the binding of ¹²⁵I-hexapeptide to rat neutrophils. The same correlation was found between these peptides in their potency to induce chemotaxis. This indicates that the rat neutrophil chemotactic receptor is like human receptor also a highly stereoselective and requires a N-formylated ligand for high affinity binding. Affinity cross-linking of ¹²⁵I-hexapeptide to rat and human neutrophil chemotactic receptor with glutaraldehyde revealed on SDS-PAGE a 85-kDa and 62-kDa major complex and a 170-kDa and 120-kDa minor complex, respectively. The 120-kDa complex was absent in human neutrophils if the cells were treated with glutaraldehyde prior to cross-linking of ¹²⁵I-hexapeptide to its receptor. Likewise, the larger complex was absent if neutrophils were exposed to heterelogous ligand (C5a) prior to glutaraldehyde treatment and cross-linking of ¹²⁵I-hexapeptide to its specific receptor. These results demonstrate that the rat neutrophils possess a functional high-affinity receptor for N-formylated chemotactic peptides and that the size of the monomeric receptor is 85-kDa and about 23-kDa larger than that of the human receptor. Upon homologous ligand binding the receptor seems to form a larger complex.     

Biochemical evidence that the type II insulin-like growth factor receptor is identical to the cation-independent mannose 6-phosphate receptor

Cloning and sequencing of the human type II insulin-like growth factor (IGF) receptor cDNA revealed an 80% deduced amino acid sequence homology with the bovine cation-independent mannose 6-phosphate (Man-6-P) receptor, suggesting identity of the two receptors (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). We have performed biochemical experiments that support this proposal. Rat liver type II IGF receptor, purified by the conventional method of IGF-II affinity chromatography, bound quantitatively to a beta-galactosidase affinity column and was eluted with Man-6-P. Bovine liver Man-6-P receptor, prepared by the conventional method of affinity chromatography on phosphomannan-Sepharose, bound IGF-II with high affinity (Kd = 1 nM). Affinity cross-linking of 125I-IGF-II to the Man-6-P receptor and analysis by sodium dodecyl sulfate-gel electrophoresis showed that beta-galactosidase, but not Man-6-P, inhibited the formation of the 250-kDa 125I-IGF-II-receptor complex. The inhibition by beta-galactosidase was prevented by coincubation with Man-6-P. 125I-IGF-II did not bind to the 46-kDa cation-dependent Man-6-P receptor. For immunologic studies we purified type II IGF receptors and Man-6-P receptors in parallel from rat placental membranes using either IGF-II- or beta-galactosidase affinity chromatography. A panel of five antisera that previously had been raised against either type II IGF receptor or Man-6-P receptor behaved identically toward type II IGF receptor versus Man-6-P receptor in ligand blocking and immunoprecipitation assays. Our data support the conclusion that the type II IGF receptor and the cation-independent Man-6-P receptor are the same protein and that the IGF-II and Man-6-P-binding sites are distinct.     

Quaternary structure of the calf testis follitropin receptor

The quaternary structural relationships between subunits of the follitropin (FSH) receptor were determined through the use of the reversible, homobifunctional, chemical cross-linking reagent bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES) after formation of covalent 125I-azidobenzoyl-FSH-receptor subunit complexes by photoaffinity labeling. This experimental approach resulted in the formation of high molecular mass complexes (116, 172, and 320 kDa) as detected by autoradiography. After reversal of the BSOCOES cross-links, a second-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of these complexes demonstrated that two lower molecular mass complexes, 64 and 84 kDa, identified previously as specific components of the FSH receptor by photoaffinity labeling alone (Smith, R. A., Branca, A. A., and Reichert, L. E., Jr. (1985) J. Biol. Chem. 260, 14297-14303) were contained within the 116- and 172-kDa complexes. In addition, the 116-kDa complex was not found to be a component of the 172-kDa complex. Since the high molecular weight complexes have molecular weights large enough to contain additional unlabeled proteins, these data indicate the possibility that there are several distinct FSH receptor subunits. Furthermore, the observation of the 320-kDa band, taken together with the observed structural relationships, suggests that the FSH receptor may contain two each of three distinct subunits with approximate molecular weights of 32,000, 48,000, and 86,000, respectively.     

Peptide mapping analysis of the avian progesterone receptor

Progesterone receptor from the chicken oviduct has been shown to exist as two 8 S forms (I and II). Form I contains a protein of Mr = 75,000 and form II contains a protein of Mr = 110,000. In addition to these hormone-binding proteins, both receptor forms contain a protein with Mr = 90,000 that does not bind steroid. To investigate the possibility that these proteins are structurally related, they were isolated by preparative sodium dodecyl sulfate gel electrophoresis and subjected to peptide mapping analyses after digestion with Staphylococcus aureus V-8 protease, papain, or alpha-chymotrypsin. Receptor proteins labeled with [32P]orthophosphate in tissue minces were also subjected to peptide mapping analysis. The electrophoretic patterns of peptide fragments of the 90-kDa protein from receptor forms I and II were identical but were different from the peptide patterns obtained from the 75- and 110-kDa proteins which generated similar peptide patterns, indicating that these are structurally related. However, some differences were evident, indicating that these latter two proteins are not identical substrates for proteases. A one-dimensional comparison of the phosphopeptide patterns from the 75- and 110-kDa proteins also showed them to be similar, but not identical. Two-dimensional maps of phosphopeptides generated from the 75- and 110-kDa protein after complete tryptic digestion revealed multiple sites of phosphorylation which were identical except for one phosphopeptide that was unique to the 110-kDa protein. These results show the two progesterone-binding proteins to be very similar in structure, but to differ considerably from the 90-kDa protein.     

Interleukin-3 stimulates the tyrosine phosphorylation of the 140-kilodalton interleukin-3 receptor

Murine interleukin-3 (mIL-3) stimulates the rapid and transient tyrosine phosphorylation of a number of proteins in mIL-3-dependent B6SUtA1 cells. Two of these proteins, p68 and p140, are maximally phosphorylated at tyrosine residues within 2 min of addition of mIL-3. Because 125I-mIL-3 can be cross-linked to both 70- and 140-kDa proteins on intact B6SUtA1 cells, we investigated whether the tyrosine phosphorylated p68 and p140 were these two mIL-3 receptor proteins. Addition of antiphosphotyrosine antibodies (alpha PTyr Abs) to cell lysates from B6SUtA1 cells, to which 125I-mIL-3 had been disuccinimidyl suberate-cross-linked, resulted in the immunoprecipitation of 125I-mIL-3 complexed to both 70- and 140-kDa proteins. To determine if the observed immunoprecipitation pattern was due to the direct interaction of alpha-PTyr Abs with these two mIL-3 receptor proteins or with tyrosine-phosphorylated proteins that were associated with the receptor proteins, cell lysates were treated with 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol, and boiled for 1 min. After removal of sodium dodecyl sulfate and 2-mercaptoethanol, alpha PTyr Abs immunoprecipitated 125I-mIL-3 cross-linked to only the 140-kDa protein. To confirm this finding, 32P-labeled B6SUtA1 cells were treated with biotinylated or fluoresceinated mIL-3. Addition of immobilized streptavidin or antifluorescein antibodies, respectively, to cell lysates from these cells resulted in the enrichment of only a 140-kDa tyrosine phosphorylated protein. Taken together, these results strongly suggest that only the 140-kDa receptor protein is tyrosine phosphorylated upon mIL-3 binding.     

Immunochemical detection of unique proteolytic fragments of the chick 1,25-dihydroxyvitamin D3 receptor. Distinct 20-kDa DNA-binding and 45-kDa hormone-binding species

We have characterized proteolytic fragments of the chick intestinal 1,25-dihyroxyvitamin D3 (1,25-(OH)2D3) receptor, produced through either exogenous or endogenous protease action, utilizing a variety of physical and functional assays coupled to immunoblot detection methodology. Treatment of intestinal cytosol with increasing concentrations of trypsin resulted in a progressive diminishment of the 60-kDa receptor concomitant with the appearance of a 20-kDa fragment reactive by Western blot analysis to an anti-1,25-(OH)2D3 receptor monoclonal antibody. Cleveland analysis supported the receptor-origin of this 20-kDa fragment: a common immunoreactive species of 12 kDa could be generated by Staphylococcus aureus V8 protease treatment of the intact 60-kDa receptor as well as the 20-kDa proteolytic product. The 20-kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60-kDa receptor and, therefore, may contain the functional DNA-binding domain of the chick 1,25-(OH)2D3 receptor. Thus, this fragment likely represents the complement of a larger hormone-bound fragment that we have previously described (Allegretto, E. A., and Pike, J.W. (1985) J. Biol. Chem. 260, 10139-10145). In contrast to the exogenous effect of trypsin, incubation of cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. Cleveland analysis was consistent with the 60-kDa receptor derivation of the 45-kDa fragment. This species retained the hormone-binding site and the antibody determinant but was devoid of DNA-binding activity. Moreover, it generated neither the trypsin-dependent 20-kDa fragment nor the V8 protease-dependent 12-kDa species and, therefore, was derived from the opposite end of the receptor molecule. These data have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the functional domains for hormone binding and DNA binding.     

Isolation of collagen binding proteins from embryonic chicken corneal epithelial cells

In the present paper, we report the isolation and characterization of embryonic corneal membrane glycoproteins that demonstrate specific affinity for collagen. Two collagen binding proteins have been isolated: a novel 70-kDa protein and a 47-kDa protein which is apparently similar to that reported by Kurkinen et al. (Kurkinen, M., Taylor, A., Garrels, J. I., and Hogan, B. (1984) J. Biol. Chem. 259, 5915-5922). Both proteins label metabolically with [35S]methionine and [3H] glucosamine. 125I iodination of cell surface proteins revealed that the two collagen binding proteins are expressed on the epithelial cell surface. The 70-kDa protein appears to be an integral membrane protein, whereas the 47-kDa protein can be removed from membranes by alkali treatment. The isolated proteins exhibit binding to native type IV collagen as well as heat-denatured type I collagen. It seems likely that we have isolated, at least in part, the cell surface receptor or receptor complex that binds collagen to the basal surface of epithelia.