Selective covalent labeling of cysteines in bovine serum albumin and in hepatoma tissue culture cell glucocorticoid receptors by dexamethasone 21-mesylate

The specificity of protein labeling by an affinity label of glucocorticoid receptors, dexamethasone 21-mesylate (Dex-Mes), was investigated using bovine serum albumin (BSA) as a model. During the early stages of [3H]Dex-Mes labeling at pH 8.8, approximately 90% of the covalent bond formation occurred at the one non-oxidized cysteine (Cys-34) of BSA. The nonspecific labeling was equally distributed over the rest of the BSA molecule. [3H]Dex-Mes labeling of Cys-34 was totally, and specifically inhibited by nearly stoichiometric amounts of the thiol-specific reagent methyl methanethiolsulfonate (MMTS). Thus both Dex-Mes and MMTS appear to react very selectively with thiols under our conditions. In reactions with hepatoma tissue culture (HTC) cell glucocorticoid receptors, MMTS was equally efficient in preventing [3H]dexamethasone binding to receptors and [3H]Dex-Mes labeling of the 98-kDa receptor protein. These results indicate that Dex-Mes labeling of the glucocorticoid receptor involves covalent reaction with at least one cysteine in the steroid binding site of the receptor. Small (approximately 1600-dalton) fragments of the [3H]Dex-Mes-labeled 98-kDa receptor were generated by limit proteolysis with trypsin, chymotrypsin, and Staphylococcus aureus V8 protease under denaturing conditions. Data from these fragments on 15% sodium dodecyl sulfate-polyacrylamide gels were consistent with all of the covalent [3H] Dex-Mes being located on one or a few cysteines in one approximately 15-residue stretch of the receptor. Further studies revealed no differences in the limit protease digestion patterns of activated and unactivated [3H]Dex-Mes-labeled receptors with trypsin, chymotrypsin, or V8 protease under denaturing conditions. These data suggest that activation does not cause any major covalent modifications of the amino acids immediately surrounding the affinity-labeled cysteine(s) of the steroid binding site.     

Affinity-labeling steroids as biologically active probes of antiglucocorticoid hormone action

The role of the glucocorticoid receptor in the expression of antiglucocorticoid action has been investigated with a chemically-reactive derivative of three glucocorticoid steroids with differing biological potencies, i.e. the C-21 mesylates of cortisol, dexamethasone and deacylcortivazol. Dexamethasone 21-mesylate (Dex-Mes) was the most useful derivative due to its favorable balance of high receptor affinity and predominantly irreversible antiglucocorticoid activity. A number of criteria have been used to conclude that [3H]Dex-Mes covalently labels glucocorticoid receptors in the steroid-binding cavity. The available data indicate that covalent Dex-Mes-labeled receptors (mol. wt approximately equal to 98,000) are responsible for the irreversible antiglucocorticoid activity while the partial agonist activity of Dex-Mes is due to non-covalent Dex-Mes-bound receptors. Further support for this hypothesis comes from the observations that deacylcortivazol 21-mesylate was a full glucocorticoid and did not affinity label receptors (and marginally labeled cytosol proteins) although it was capable of covalently-labeling bovine serum albumin. Several mechanisms for the expression of irreversible antiglucocorticoid activity by covalent Dex-Mes-labeled receptors were examined and can be eliminated. Covalent receptor-Dex-Mes complexes formed in whole HTC cells were found to have a decreased capacity for nuclear binding. This decreased nuclear-binding capacity could be responsible for the whole-cell irreversible antiglucocorticoid activity of Dex-Mes.     

Identification of hormone-interacting amino acid residues within the steroid-binding domain of the glucocorticoid receptor in relation to other steroid hormone receptors

Purified rat liver glucocorticoid receptor was covalently charged with [3H]glucocorticoid by photoaffinity labeling (UV irradiation of [3H]triamcinolone acetonide-glucocorticoid receptor) or affinity labeling (incubation with [3H]dexamethasone mesylate). After labeling, separate samples of the denatured receptor were cleaved with trypsin (directly or after prior succinylation), chymotrypsin, and cyanogen bromide. Labeled residues in the peptides obtained were identified by radiosequence analysis. The peaks of radioactivity corresponded to Met-622 and Cys-754 after photoaffinity labeling with [3H]triamcinolone acetonide and Cys-656 after affinity labeling with [3H]dexamethasone mesylate. The labeled residues are all positioned within hydrophobic segments of the steroid-binding domain. The patterns of hydropathy and secondary structure for the glucocorticoid receptor are highly similar to those for the progestin receptor and similar but less so to those for the estrogen receptor and to those for c-erb A.     

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.     

Phosphoenolpyruvate carboxylase of Escherichia coli K-12. N- and C-terminal sequences and tentative assignment of the catalytically essential cysteine residue

The N- and C-terminal amino acid sequences of phosphoenolpyruvate carboxylase [EC 4.1.1.31] from Escherichia coli K-12 were determined to establish the primary structure deduced from the nucleotide sequence of the cloned gene for the enzyme (Fujita, N., Miwa, T., Ishijima, S., Izui, K., & Katsuki, H. (1984) J. Biochem. 95, 909-916). As predicted from the nucleotide sequence, two polypeptides were produced upon treatment with hydroxylamine, which specifically cleaves the Asn-Gly bond, and their amino acid compositions were also in accordance with those predicted. The tryptic peptides which contained cysteine residues labeled with a fluorescent reagent, N-[7-(dimethylamino)-4-methylcoumarinyl]maleimide, were isolated by high-performance liquid chromatography and partially sequenced. All of them could be assigned on the deduced primary structure. The modified cysteine residues were Cys-157, Cys-385, Cys-458, Cys-568, Cys-665, and Cys-754. Furthermore, the essential cysteine residue which is presumably located at or near the active site was tentatively identified as Cys-568, since it was consistently protected against the modification by 2-phospholactate, a substrate analog.     

Identification of cysteine-644 as the covalent site of attachment of dexamethasone 21-mesylate to murine glucocorticoid receptors in WEHI-7 cells

Dexamethasone 21-mesylate is a highly specific synthetic glucocorticoid derivative that binds covalently to glucocorticoid receptors via sulfhydryl groups. We have identified the amino acid that reacts with the dexamethasone 21-mesylate by using enzymatic digestion and microsequencing for radiolabel. Nonactivated glucocorticoid receptors obtained from labeling intact WEHI-7 mouse thymoma cells with [3H]dexamethasone 21-mesylate were immunopurified and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified approximately 100-kDa steroid-binding subunit was eluted from gel slices and subjected to enzymatic digestion. Trypsin digestion followed by reversed-phase high-performance liquid chromatography (reversed-phase HPLC) produced a single [3H]dexamethasone 21-mesylate labeled peptide. Automated Edman degradation of this peptide revealed that the [3H]dexamethasone 21-mesylate was located at position 5 from the amino terminus. Dual-isotope labeling studies with [3H]dexamethasone 21-mesylate and [35S]methionine demonstrated that this peptide contained methionine. Staphylococcus aureus V8 protease digestion of [3H]dexamethasone 21-mesylate labeled steroid-binding subunits generated a different radiolabeled peptide containing label at position 7 from the amino terminus. On the basis of the published amino acid sequence of the murine glucocorticoid receptor, our data clearly identify cysteine-644 as the single residue in the steroid-binding domain that covalently binds dexamethasone 21-mesylate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of glucocorticoid receptor in HeLa-S3 cells

Glucocorticoid receptor of the human cell line HeLa-S3 has been characterized and has been compared to rat and to mouse glucocorticoid receptors. If HeLa cells were lysed in the absence of glucocorticoid, glucocorticoid receptor was isolated in a nonactivated form, which did not bind to DNA-cellulose. If HeLa cells were preincubated with glucocorticoid, glucocorticoid receptor was isolated in an activated, DNA-binding form. HeLa cell glucocorticoid receptor bound [3H]triamcinolone acetonide with a dissociation constant (KD = 1.3 nM at 0 degrees C) that was similar to those of mouse and rat glucocorticoid receptors. Similarly, the relative binding affinities for steroid hormones decreased in the order of triamcinolone acetonide greater than dexamethasone greater than promegestone greater than methyltrienolone greater than aldosterone greater than or equal to moxestrol. Nonactivated and activated receptors were characterized by high-resolution anion-exchange chromatography (FPLC), DNA-cellulose chromatography, and sucrose gradient centrifugation. Human, mouse, and rat nonactivated glucocorticoid receptors had very similar ionic and sedimentation properties. Activated glucocorticoid receptors were eluted at similar salt concentrations from DNA-cellulose columns but at different salt concentrations from the FPLC column. A monoclonal mouse anti-rat liver glucocorticoid receptor antibody [Westphal, H.M., Mugele, K., Beato, M., & Gehring, U. (1984) EMBO J. 3, 1493-1498] did not cross-react with HeLa cell glucocorticoid receptor. Glucocorticoid receptors of HeLa, HTC, and S49.1 cells were affinity labeled with [3H]dexamethasone and with [3H]dexamethasone 21-mesylate. The molecular weights of [3H]dexamethasone 21-mesylate labeled glucocorticoid receptors (MT 96 000 +/- 1000) were undistinguishable by polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of cysteines 640, 656, and 661 in steroid binding to rat glucocorticoid receptors.

The involvement of a vicinally spaced dithiol group in steroid binding to the glucocorticoid receptor has been deduced from experiments with the thiol-specific reagent methyl methanethiolsulfonate and the vicinal dithiol-specific reagent sodium arsenite. The vicinally spaced dithiol appears to reside in the 16-kDa trypsin fragment of the receptor, which is thought to contain 3 cysteines (Cys-640, -656, and -661 of the rat receptor) and binds hormone with an approximately 23-fold lower affinity than does the intact 98-kDa receptor. We now report that the steroid binding specificity of preparations of this 16-kDa fragment and the intact receptor are virtually identical. This finding supports our designation of the 16-kDa fragment as a steroid-binding core domain and validates our continued use of this tryptic fragment in studies of steroid binding. To identify the cysteines which comprise the vicinally spaced dithiol group, and to examine further the role of cysteines in steroid binding, a total of five point mutant receptors were prepared: cysteine-to-serine for each suspected cysteine, cysteine-to-glycine for Cys-656, and the C656,661S double mutant. Unexpectedly, each receptor with a single point mutation still bound steroid. Even the double mutant (C656,661S) bound steroid with wild type affinity. These results suggest that none of these cysteines are directly required either for steroid binding to the glucocorticoid receptor or for heat shock protein 90 association with the receptor. However, the presence of Cys-656 was obligatory for covalent labeling of the receptor by [3H]dexamethasone 21-mesylate. Studies with preparations of the 98 and 16 kDa forms of these mutant receptors revealed both that Cys-656 and -661 comprise the vicinally spaced dithiols reacting with arsenite and that any two of the three thiols could form an intramolecular disulfide after treatment with low concentrations of methyl methanethiolsulfonate. These data, in conjunction with those from experiments on the effects of steric bulk on various receptor functions, support a model for the ligand binding cavity of the receptor that involves all three thiols in a flexible cleft but where thiol-steroid interactions are not essential for binding.     

Identification of the arylazido-beta-alanyl-NAD+-modified site in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry

We have identified the site labeled by arylazido-beta-alanyl-NAD+ (A3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)NAD+) in rabbit muscle glyceraldehyde-3-phosphate dehydrogenase by microsequencing and fast atom bombardment mass spectrometry. This NAD+ photoaffinity analogue has been previously demonstrated to modify glyceraldehyde-3-phosphate dehydrogenase in a very specific manner and probably at the active site of the enzyme [Chen, S., Davis, H., Vierra, J. R., & Guillory, R. J. (1984) Biochem. Biophys. Stud. Proteins Nucleic Acids, Proc. Int. Symp., 3rd, 407-425]. The label is associated exclusively with a tryptic peptide that has the sequence Ile-Val-Ser-Asn-Ala-Ser-Cys-Thr-Thr-Asn. In comparison to the amino acid sequence of glyceraldehyde-3-phosphate dehydrogenase from other species, this peptide is in a highly conserved region and is part of the active site of the enzyme. The cysteine residue at position seven was predominantly labeled and suggested to be the site modified by arylazido-beta-alanyl-NAD+. This cysteine residue corresponds to the Cys-149 in the pig muscle enzyme, which has been shown to be an essential residue for the enzyme activity. The present investigation clearly demonstrates that arylazido-beta-alanyl-NAD+ is a useful photoaffinity probe to characterize the active sites of NAD(H)-dependent enzymes.     

Evidence for a signal sequence at the N terminus of the common precursor to adrenocorticothrophin and beta-lipotropin in mouse pituitary cells

The precursor to corticotropin and beta-endorphin was synthesized in a reticulocyte cell-free system under the direction of mRNA from mouse AtT-20 pituitary tumor cells in the presence of [3H]proline, [3H]phenylalanine, [3H]leucine, [3H]valine, [3H]isoleucine or [35S]methionine. Automatic Edman degradation of the radioactive cell-free product showed the following N-terminal sequence: Pro-1, Met-2, Leu-11, Leu-12, Leu-13, Leu-15, Leu-16, Leu-17, Ile-21 and Val-23. The corticotropin-endorphin precursor was also labeled in AtT-20 cells with [3H]valine, [3H]leucine, [3H]tryptophan, [3H]serine, [35S]methionine or [35S]cysteine. Automatic Edman degradation of the radioactive intact cell form gave the following N-terminal sequence: Trp-1, Cys-2, Leu-3, Ser-5, Ser-6, Val-7, Cys-8, Leu-11, Leu-17, Leu-18 and tentatively Met-27. The sequence of the intact cell form from AtT-20 cells matches the sequence of the cell-free form of bovine pituitary precursor beginning at Trp-27, as determined by recombinant DNA technology [Nakanishi, S., Inoue, A., Kita, T., Nakamura, M., Chang, A. C. Y., Cohen, S. N., and Numa, S. (1979) Nature (Lond.) 278, 423-427]. The sequence of the mouse pituitary mRNA-directed cell-free translation product also matches the bovine precursor beginning at Pro-2. The results suggest that both the mouse and bovine precursors possess a signal sequence of 26 amino acids which is cleaved in intact cells. CNBr cleavage of [35S]cysteine-labelled intact cell precursor gave rise to an N-terminal fragment of a size compatible with the presence of a methionyl residue at or near position 27.     

Radiosequence analysis of the human progestin receptor charged with [3H]promegestone. A comparison with the glucocorticoid receptor

Partially purified preparations of the human progestin receptor and the human and rat glucocorticoid receptor proteins were covalently charged with the synthetic progestin, [3H]promegestone, by photoaffinity labeling. After labeling, the denaturated protein was cleaved and the mixture of peptides subjected to radiosequence analysis as previously described for the rat glucocorticoid receptor protein (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). The radioactivity labels identified, corresponded to Met-759 and Met-909 after photoaffinity labeling of the human progestin receptor, and Met-622 and Cys-754 after labeling of the rat glucocorticoid receptor. The residues labeled in the glucocorticoid receptor are the same as those previously reported to bind triamcinolone actonide. The corresponding residues were also labeled in the human glucocorticoid receptor. Met-759 of the progestin receptor and Met-622 of the rat glucocorticoid receptor are positioned within a segment with an overall high degree of sequence similarity and are equivalent. However, Met-909 (progestin receptor) and Cys-754 (glucocorticoid receptor) do not occur within equivalent segments of the two proteins. Thus, although the two classes of steroid hormone share a common structure within the A-ring, there are subtle differences in their interaction with the two separate receptor proteins.     

Identification of cysteine 530 as the covalent attachment site of an affinity-labeling estrogen (ketononestrol aziridine) and antiestrogen (tamoxifen aziridine) in the human estrogen receptor

Radiosequence analysis of peptide fragments of the estrogen receptor (ER) from MCF-7 human breast cancer cells has been used to identify cysteine 530 as the site of covalent attachment of an estrogenic affinity label, ketononestrol aziridine (KNA), and an antiestrogenic affinity label, tamoxifen aziridine (TAZ). ER from MCF-7 cells was covalently labeled with [3H]TAZ or [3H]KNA and purified to greater than 95% homogeneity by immunoadsorbent chromatography. Limit digest peptide fragments, generated by prolonged exposure of the labeled receptor to trypsin, cyanogen bromide, or Staphylococcus aureus V8 protease, were purified to homogeneity by high performance liquid chromatography (HPLC), and the position of the labeled residue was determined by sequential Edman degradation. With both aziridines, the labeled residue was at position 1 in the tryptic peptide, position 2 in the cyanogen bromide peptide, and position 7 in the V8 protease peptide. This localizes the site of labeling to a single cysteine at position 530 in the receptor sequence. The identity of cysteine as the site of labeling was confirmed by HPLC comparison of the TAZ-labeled amino acid (as the phenylthiohydantoin and phenylthiocarbamyl derivatives) and the KNA-labeled amino acid (as the phenylthiocarbamyl derivative) with authentic standards prepared by total synthesis. Cysteine 530 is located in the hormone binding domain of the receptor, near its carboxyl terminus. This location is consistent with earlier studies using sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze the size of the proteolytic fragments containing the covalent labeling sites for TAZ and KNA and the antigen recognition sites for monoclonal antibodies. The fact that both the estrogenic and antiestrogenic affinity labeling agents react covalently with the same cysteine indicates that differences in receptor-agonist and receptor-antagonist complexes do not result in differential covalent labeling of amino acid residues in the hormone binding domain.     

The interaction site for tamoxifen aziridine with the bovine estrogen receptor

Calf uterine estrogen receptor was covalently labeled with [3H]tamoxifen aziridine during affinity chromatography purification. After carboxymethylation, affinity labeled receptor was digested with trypsin under limit conditions and the labeled peptides were fractionated by reversed-phase high performance liquid chromatography into one major and two minor components. Sequence analysis of the dominant labeled fragment indicated the facile cleavage of label during Edman degradation but identified two peptides, both derived from the extreme carboxyl terminus of the steroid-binding domain. The 17 residues of one peptide were fully conserved in all estrogen receptors. This fragment contained five nucleophilic amino acids and was considered as the more favored interaction site for tamoxifen aziridine. A corresponding region of the glucocorticoid receptor has recently been identified as one of three major contact sites for glucocorticoids (Carlstedt-Duke, J., Str?mstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and J?rnvall, H. (1988) J. Biol. Chem. 263, 6842-6846). A comparison of amino acid physical characteristics in the hormone-binding domains of human estrogen and glucocorticoid receptors demonstrated an excellent structural correlation between the two regions and delineated elements in the estrogen receptor which may be directly involved in estradiol binding.     

Steroid binding to hepatoma tissue culture cell glucocorticoid receptors involves at least two sulfhydryl groups

The presence of a thiol in the steroid binding cavity of glucocorticoid receptors has recently been proved by our affinity labeling of Cys-656 in the steroid binding domain of rat receptors (Simons, S. S., Jr., Pumphrey, J. G., Rudikoff, S., and Eisen, H. J. (1987) J. Biol. Chem. 262, 9676-9680). Studies with the sterically small, thiol-specific reagent methyl methanethiolsulfonate (MMTS) now reveal the involvement of at least two sulfhydryl groups in steroid binding. While the dose-response curves for [3H]dexamethasone binding versus thiol reagent are normally sigmoidal, an unusual bimodal curve is obtained with MMTS in which dexamethasone binding is eliminated at low, but maintained at intermediate, MMTS concentrations. This bimodal dose-response curve demands the involvement of two (or more) thiol groups. Those receptors pretreated with intermediate concentrations of MMTS retain approximately 70% of the initial binding capacity and one-fifth the affinity for dexamethasone. Solutions of this low affinity form of receptor contain essentially no accessible -SH groups, and all of the usual covalent labeling by dexamethasone 21-mesylate of various proteins, including the receptor, is blocked. The facts, that this low affinity form of the receptor is not affected by added iodoacetamide, cannot be produced from the nonsteroid binding form of receptor simply by adding more MMTS, and displays different kinetics of formation than does the nonsteroid binding form of receptor all argue that reaction of the receptor with intermediate and low MMTS, concentrations occurs via different pathways. Nevertheless, the effects of both concentrations of MMTS on the receptor are fully reversible with added dithiothreitol. The kinetics of inhibition of [3H]dexamethasone binding at low MMTS concentrations are independent of receptor concentration, indicating an intramolecular reaction. Collectively these data suggest a model of steroid binding involving two thiols, one of which appears to be Cys-656. Low concentrations of MMTS induce the formation of an intramolecular disulfide, which prevents steroid binding, while the intermediate MMTS concentrations convert both thiols directly to mixed disulfides, and steroid binding persists. Thus, reduced thiols do not appear to be required for steroid binding if the steric bulk of the oxidized thiols is small.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Site-directed mutagenesis suggests close functional relationship between a human rhinovirus 3C cysteine protease and cellular trypsin-like serine proteases

Human rhinoviruses, like other picornaviruses, encode a cysteine protease (designated 3C) which cleaves mainly at viral Gln-Gly pairs. There are significant areas of homology between picornavirus 3C cysteine proteases and cellular serine proteases (e.g. trypsin), suggesting a functional relationship between their catalytic regions. To test this functional relationship, we made single substitutions in human rhinovirus type 14 protease 3C at seven amino acid positions which are highly conserved in the 3C proteases of animal picornaviruses. Substitutions at either His-40, Asp-85, or Cys-146, equivalent to the trypsin catalytic triad His-57, Asp-102, and Ser-195, respectively, completely abolished 3C proteolytic activity. Single substitutions were also made at either Thr-141, Gly-158, His-160, or Gly-162, which are equivalent to the trypsin specificity pocket region. Only the mutant with a conservative Thr-141 to Ser substitution exhibited proteolytic activity, which was much reduced compared with the parent. These results, together with immunoprecipitation data which indicate that Asp-85, Thr-141, and Cys-146 lie in accessible surface regions, suggest that the catalytic mechanism of picornavirus 3C cysteine proteases is closely related to that of cellular trypsin-like serine proteases.     

Extracellular release as the major degradative pathway of the insulin-like growth factor II/mannose 6-phosphate receptor.

The presence of a soluble, truncated form of the IGF-II/Man-6-P receptor in serum has suggested that cleavage from the cell surface may be an initial step in the degradation of this protein (MacDonald, R. G., Tepper, M. A., Clairmont, K. B., Perregaux, S. B., and Czech, M. P. (1989) J. Biol. Chem. 264, 3256-3261). In order to test this hypothesis, we pulse-labeled cultured BRL-3A rat liver cells with [35S]methionine and [35S]cysteine and measured the fate of labeled receptor at various times after incubation with unlabeled amino acids. It was found that the appearance of labeled IGF-II/Man-6-P receptor in the medium accounts quantitatively for the loss of labeled receptor from the BRL-3A cells. In similar experiments with Chinese hamster ovary cells, L6 rat myoblasts, and chick embryo fibroblasts, labeled receptor from the cell membranes decreases with a time course corresponding to the appearance of soluble receptor in the medium. The release of labeled receptor into the medium can be blocked by the addition of the protease inhibitors aprotinin, chymostatin, or phenylmethylsulfonyl fluoride, but not antipain, leupeptin, and benzamidine. The results are consistent with the hypothesis that the degradation and loss of cellular IGF-II/Man-6-P receptors occurs by a nonlysosomal mechanism involving their proteolysis and removal into the extracellular fluid.     

Mapping of the acetylcholine binding site of the nicotinic acetylcholine receptor: [3H]nicotine as an agonist photoaffinity label

The agonist [3H]nicotine was used as a photoaffinity label for the acetylcholine binding sites on the Torpedo nicotinic acetylcholine receptor (AChR). [3H]nicotine binds at equilibrium with Keq = 0.6 microM to the agonist binding sites. Irradiation with 254-nm light of AChR-rich membranes equilibrated with [3H]nicotine resulted in covalent incorporation into the alpha- and gamma-subunits, which was inhibited by agonists and competitive antagonists but not by noncompetitive antagonists. Inhibition of labeling by d-tubocurarine demonstrated that the alpha-subunit was labeled via both agonist sites but the gamma-subunit was labeled only via the site that binds d-tubocurarine with high affinity. Within the alpha-subunit, 93% of the labeling was contained within a 20-kDa Staphylococcus aureus V8 proteolytic fragment beginning at Ser-173. Sequence analysis of this peptide indicated that approximately 80% of the incorporation was into Tyr-198, approximately 13% was into Cys-192, and approximately 7% was into Tyr-190. Chymotryptic digestion of the alpha-subunit confirmed that Tyr-198 was the principal amino acid labeled by [3H]nicotine. This confirmation required a novel radio-sequencing strategy employing omicron-phthalaldehyde, since the efficiency of photolabeling was low (approximately 1.0%) and the labeled chymotryptic peptide was not isolated in sufficient quantity to be identified by mass. [3H]Nicotine, which is the first photoaffinity agonist used, labels primarily Tyr-198 in contrast to competitive antagonist affinity labels, which label primarily Tyr-190 and Cys-192/Cys-193.