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PubMed1991

Detection of the Ah receptor in rainbow trout: use of 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin in cell culture.

The Ah receptor was detected in RTG-2 cells (rainbow trout embryonic gonad cells) following the addition of the photoaffinity ligand, [125I]2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, to cells in culture. Cytosolic and nuclear extracts were prepared and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and revealed one radiolabeled band. Very little non-specific binding was observed under the conditions employed when compared to photoaffinity labeling RTG-2 cytosolic extracts in vitro. The photoaffinity-labeled Ah receptor in RTG-2 cytosol was analyzed by sucrose density centrifugation. The cytosolic form was observed to sediment at approximately 9.8S and the high salt nuclear extract form at approximately 7.5S. The relative molecular weight of the Ah receptor was determined to be 145 kDa under denaturing conditions and is considerably larger than the Ah receptor from mammalian sources. Inhibition of photoaffinity ligand binding to the RTG-2 cytosolic Ah receptor by competing ligands revealed the same rank order of ligand affinity as that previously demonstrated for the mouse Ah receptor.

Affinity Labels↗
PubMed1989

Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression.

HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2006

The calcium channel blockers, 1,4-dihydropyridines, are substrates of the multidrug resistance-linked ABC drug transporter, ABCG2.

The human ATP-binding cassette transporter, ABCG2, confers resistance to multiple chemotherapeutic agents and also affects the bioavailability of different drugs. [(125)I]Iodoarylazidoprazosin (IAAP) and [(3)H]azidopine were used for photoaffinity labeling of ABCG2 in this study. We show here for the first time that both of these photoaffinity analogues are transport substrates for ABCG2 and that [(3)H]azidopine can also be used to photolabel both wild-type R482-ABCG2 and mutant T482-ABCG2. We further used these assays to screen for potential substrates or modulators of ABCG2 and observed that 1,4-dihydropyridines such as nicardipine and nifedipine, which are clinically used as antihypertensive agents, inhibited the photolabeling of ABCG2 with [(125)I]IAAP and [(3)H]azidopine as well as the transport of these photoaffinity analogues by ABCG2. Furthermore, [(3)H]nitrendipine and bodipy-Fl-dihydropyridine accumulation assays showed that these compounds are transported by ABCG2. These dihydropyridines also inhibited the efflux of the known ABCG2 substrates, mitoxantrone and pheophorbide-a, from ABCG2-overexpressing cells, and nicardipine was more potent in inhibiting this transport. Both nicardipine and nifedipine stimulated the ATPase activity of ABCG2, and the nifedipine-stimulated activity was inhibited by fumitremorgin C, suggesting that these agents might interact at the same site on the transporter. In addition, nontoxic concentrations of dihydropyridines increased the sensitivity of ABCG2-expressing cells to mitoxantrone by 3-5-fold. In aggregate, results from the photoaffinity labeling and efflux assays using [(125)I]IAAP and [(3)H]azidopine demonstrate that 1,4-dihydropyridines are substrates of ABCG2 and that these photolabels can be used to screen new substrates and/or inhibitors of this transporter.

ATP Binding Cassette Transporter, Subfamily G, Mem↗
PubMed1976

Chemical probes of estrogen action: affinity labeling.

A systemic approach has been taken in the preparation and evaluation of photoaffinity labeling agents for the estrogen receptor from rat and lamb uterus. Several derivatives of estradiol and the nonsteroidal estrogen, hexestrol, containing photoreactive diazocarbonyl or azide functions have been synthesized. The receptor binding affinity of these compounds and their capacity to photointeract with the estrogen binding site (inactivate) can be assayed inderectly by competition assays. Several of the compounds that showed both resonably high binding affinities and inactivation efficiencies have been prepared in high specific activity, tritium-labeled form. Direct binding measurements with these derivatives in unpurified rat uterine receptor preparations, show that while these compounds bind to the receptor, they also show considerable nonspecific binding to nonreceptor proteins. Irradiation of these derivatives in rat uterine cytosol preparations results in incorporation of large amounts of radioactivity into protein in a covalent fashion. The amount of nonspecific labeling is so large, however, that estrogen site specificity (indicated by protection with unlabeled estradiol) cannot be demonstrated. More recently, we have used a partially purified receptor preparation from lamb uterus. The receptor in this preparation has been disaggregated by mild trypsinization and can be electrophoresed in native form. Electrophoretic analysis of the proteins in photolabeled preparations show some covalent incorporation into the receptor region of the gel with one derivative but not with another. The effectiveness of the photoaffinity labeling reagents prepared thus far is assessed, and suggestions are made for the design of new, more effective reagents.

Affinity Labels↗
PubMed2005

Probing the reorganization of the nicotinic acetylcholine receptor during desensitization by time-resolved covalent labeling using [3H]AC5, a photoactivatable agonist.

The structural reorganizations occurring on the nicotinic acetylcholine receptor (nAChR) during activation and subsequent desensitization have been investigated through time-resolved photoaffinity labeling using a photoactivatable nicotinic agonist. [(3)H]AC5 is a photosensitive nicotinic probe with high affinity for the desensitized state of the Torpedo marmorata receptor (K(D) = 5 nM) that displays full agonist activity on the Torpedo californica receptor expressed in oocytes (EC(50) = 1.2 microM). Photoaffinity labeling of this receptor in the desensitized state showed a predominant specific labeling of gamma and delta subunits, whereas the alpha subunit was barely labeled. Using a stopped-flow device combined with a flash photolysis quenching system, we investigated the covalent mapping of the subunits as a function of incubation time of the receptor with [(3)H]AC5 (17 ms-1.25 h). During agonist-induced desensitization, specific labeling increased substantially, with similar time constants for gamma and delta subunits (0.016 s(-1)), whereas labeling of the alpha subunit remained relatively low. Therefore, the repartition of radioactivity shifted during desensitization from a weak but predominant labeling of the alpha and gamma subunits toward a substantial labeling of gamma and delta subunits. The observed time-dependent labeling pattern together with AC5 docking into a homology model of the T. californica nAChR suggest a subunit reorganization during agonist-induced desensitization, leading to a tightly packed arrangement that corresponds to a stable high affinity state for agonists.

Animals↗
PubMed1976

Monovalent derivatives of concanavalin A.

Monovalent dimers of concanavalin A (Con A) have been prepared by a combination of succinylation and photoaffinity labeling. Partial derivatization of native Con A using the photoaffinity label, p-azidophenyl-alpha-D-mannopyranoside, followed by affinity chromatography yielded a fraction that consisted of dimers with a single saccharide-binding site at pH 5. These monovalent dimers formed divalent tetramers at pH 7. In order to achieve a monovalent dimer at this pH, the divalent tetramers were succinylated by previously developed methods. Ultracentrifugation, equilibrium dialysis, and chromatographic experiments indicated that the resultant preparations consisted mainly of monovalent dimers which showed subunit exchange to yield about 15% divalent dimers after 12 hr at physiological pH. Freshly prepared material failed to agglutinate sheep erythrocytes at concentrations 500-fold higher than native tetravalent Con A. In addition, they showed saturating dose-response curves of mitogenic stimulation of mouse splenic lymphocytes. These curves resembled those of divalent succinyl-Con A but not those of the native molecule. Further development of methods for preparing stable monovalent derivatives of Con A should allow a refined analysis of the effects of lectin valence at the cell surface.

Affinity Labels↗
PubMed1998

Analysis of random recombination between human MDR1 and mouse mdr1a cDNA in a pHaMDR-dihydrofolate reductase bicistronic expression system.

Human P-glycoprotein (Pgp) confers multidrug resistance (MDR) to otherwise sensitive cells. The homologous mouse Pgps, which are encoded by mouse mdr1a (also known as mdr3) and mdr1b (also known as mdr1), confer different degrees of resistance to the same MDR drugs and inhibitors. To create recombinants for the study of sequences responsible for these differences in drug-resistance, chimeric cDNA libraries can be constructed by homologous recombination of pools of related sequences. This mutagenesis approach is called DNA shuffling. To select for chimeric Pgp with an altered resistance profile, DNA shuffling between the homologous but not identical drug interacting transmembrane domains 5 and 6 of human MDR1 and mouse mdr1a was used. The chimeric proteins were expressed in human KB-3-1 cells. One recombinant Pgp (clone 3-4) with a novel phenotype was analyzed in detail. Inhibitors of Pgp, including verapamil and cyclosporin A, were less effective in reversing resistance of the chimeric Pgp compared with wild-type Pgp, for certain drugs. However, [125I]iodoarylazidoprazosin photoaffinity labeling of the chimeric Pgp and its binding competition with cyclosporin A, showed that cyclosporin A competed for the photoaffinity labeling. The chimeric Pgp cells stained less well with human-specific anti-Pgp mAb MRK16 than wild-type Pgp, despite having the described epitopes for MRK16. Staining with human-specific mAb UIC2 was increased when the chimeric protein was compared with wild-type Pgp. These results suggest an alteration in exposure of human Pgp specific epitopes in this chimeric Pgp, as well as a change in the interaction of reversing agents with the chimeric protein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Functional reassembly of ATP-dependent xenobiotic transport by the N- and C-terminal domains of RLIP76 and identification of ATP binding sequences.

We have recently shown that RLIP76, a Ral-binding, GTPase-activating protein, is an ATP-dependent transporter of doxorubicin (DOX) as well as glutathione conjugates [Awasthi, S., et al. (2000) Biochemistry 39, 9327-9334]. RLIP76 overexpressed in human cells or transformed E. coli undergoes proteolysis to yield several fragments, including two prominent peptides, N-RLIP76(1-367) and C-RLIP76(410-655), from the N- and C-terminal domains, respectively. To investigate whether the fragmentation of RLIP76 has any relevance to its transport function, we have studied the characteristics of these two peptide fragments. Recombinant N-RLIP76(1-367) and C-RLIP76(410-655) were purified from overexpressing transformed E. coli. While N-RLIP76(1-367) readily underwent proteolysis, showing SDS-gel patterns similar to those of RLIP76, C-RLIP76(410-655) was resistant to such degradation. Both N-RLIP76(1-367) and C-RLIP76(410-655) had ATPase activity (K(m) for ATP, 2.5 and 2.0 mM, respectively) which was stimulated by DNP-SG, DOX, and colchicine (COL). ATP binding to both peptides was confirmed by photoaffinity labeling with 8-azido-ATP that was increased in the presence of compounds that stimulated their ATPase activity. Photoaffinity labeling was also increased in the presence of vanadate, indicating trapping of a reaction intermediate in the ATP binding site. The ATP binding sites in N-RLIP76(1-367) and C-RLIP76(410-655) were identified to be (69)GKKKGK(74) and (418)GGIKDLSK(425), respectively. Mutation of K(74) and K(425) to M residues, in N-RLIP76(1-367) and C-RLIP76(410-655), respectively, abrogated their ATPase activity as well as azido-ATP labeling. Proteoliposomes reconstituted with either N-RLIP76(1-367) or C-RLIP76(410-655) alone did not catalyze ATP-dependent transport of DOX or COL. However, proteoliposomes reconstituted with a mixture of N-RLIP76(1-367) and C-RLIP76(410-655) mediated such transport. Proteoliposomes reconstituted with the mixture of mutant peptides lacking ATPase activity did not exhibit transport activity. Present studies have identified the ATP binding sites in RLIP76, and show that DOX and COL transport can be reconstituted by two fragments of RLIP76.

ATP-Binding Cassette Transporters↗
PubMed1984

Short-wave ultraviolet irradiation increases photo-affinity labeling of benzodiazepine sites.

Photoaffinity labeling of benzodiazepine sites with [3H]-flunitrazepam was examined using either long-wave (366 nm) or short-wave (254 nm) ultraviolet irradiation. A multiple exposure protocol was employed so that the time course of the process could be determined as well as the fraction of total sites labeled. At 366 nm, approximately 20% of the total sites present were labeled and the remainder showed reduced affinity for flunitrazepam, in agreement with published reports. When membranes from either cortex or cerebellum were irradiated at 254 nm, however, the fraction of sites labeled increased above 40%. The change in the ratio of labeled sites to those showing reduced affinity, as well as differences in the time courses of the two phenomena, are taken as evidence that the labeling and affinity change are independent processes rather than two effects of a single event. Inhibition of labeling by clonazepam and Ro15-1788 indicated the "central" nature of the labeled sites.

Affinity Labels↗
PubMed1988

Effects of indole alkaloids on multidrug resistance and labeling of P-glycoprotein by a photoaffinity analog of vinblastine.

Multidrug resistant cells are characterized by decreased drug accumulation and retention, thought to be mediated by a high molecular weight glycoprotein, P-glycoprotein (P-gp). Agents such as verapamil have been shown to increase anticancer drug cytotoxicity and increase the amount of drug accumulated and retained by such cells. We show here that in addition to verapamil, reserpine, chloroquine, quinine, quinacrine, yohimbine, vindoline, and catharanthine also enhance the cytotoxicity of vinblastine (VLB) in a multidrug resistant, human leukemic cell line, CEM/VLB1K, described here for the first time. These cells express P-gp as a doublet that is photoaffinity labeled by the analog of VLB, N(p-azido-[3-125I]salicyl)-N'-beta-aminoethylvindesine ([125I]NASV). Both reserpine and, to a lesser extent, verapamil, compete with [125I]NASV for binding to P-gp. We also found that chloroquine, quinacrine, vindoline, and catharanthine, each of which enhanced VLB cytotoxicity in CEM/VLB1K cells by 10- to 15-fold, similarly inhibited [125I]NASV labeling of P-gp. However, neither quinine nor yohimbine inhibited this labeling, and the inhibition produced by catharanthine and vindoline was the greatest or exclusively on the lower band of the P-gp doublet. Our results suggest a complex relationship between the ability of a compound to modulate MDR and its ability to compete for binding to P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Thyroid hormone export in rat FRTL-5 thyroid cells and mouse NIH-3T3 cells is carrier-mediated, verapamil-sensitive, and stereospecific.

Export of L-T3 out of the cell is one factor governing the cellular T3 content and response. We previously observed in liver-derived cells that T3 export was inhibited by verapamil, suggesting that it is due to either ATP-binding cassette/multidrug resistance (MDR1/mdr1b) or multidrug resistance-related (MRP1/mrp1) proteins. To test this hypothesis we measured T3 export in FRTL-5, NIH-3T3, and rat hepatoma (HTC) cells that varied in expression of these proteins. FRTL-5 and NIH-3T3 cells were found to contain a T3 efflux mechanism that is verapamil inhibitable, saturable, and stereospecific. By contrast, T3 efflux in HTC cells was slow and unaffected by verapamil. Neither FRTL-5 nor NIH-3T3 cells express mdrlb, but all three cell types express mrpl, as assessed by immunoblotting. Overexpression of MDR1 in NIH-3T3 cells did not enhance verapamil-inhibitable T3 efflux. Photoaffinity labeling of FRTL-5 and NIH-3T3 cells with [125I]L-T3 revealed a labeled 90- to 100-kDa protein that was not present in HTC cells. Verapamil and excess nonradioactive L-T3, but not D-T3, inhibited labeling of this protein. The lack of correlation between T3 efflux and MDR1 and mrpl expression and the finding of a photoaffinity-labeled putative transport protein smaller than MDR1 or mrp1 protein (approximately 170 kDa) suggest that a novel protein is involved in the transport of T3 out of cells.

3T3 Cells↗
PubMed2004

Key differences in molecular complexes of the cholecystokinin receptor with structurally related peptide agonist, partial agonist, and antagonist.

The molecular basis of docking of receptor ligands having differences in biological activity and their subsequent effects on receptor conformation represent areas of great interest. In this work, we focus on the sulfated tyrosyl residue in position 27 of cholecystokinin (CCK) and its spatial approximation with the type A CCK receptor residue Arg(197) that has been predicted from mutagenesis experiments. We have examined the requirement for sulfation of this residue in a series of structurally related peptide agonists, partial agonists, and antagonists using assays of receptor binding and biological activity. Whereas sulfation of CCK position 27 was critical for affinity and potency of a full agonist, it had progressively less effect as the biological activity of the ligand was reduced. It had an intermediate effect on the partial agonist and no effect on the antagonist. In addition, photoaffinity labeling was used to determine the spatial approximations between the receptor and residue 27 of the agonist and antagonist in this series. Direct photoaffinity labeling with a full agonist probe confirmed the spatial approximation of ligand residue 27 and receptor residue Arg(197) in the active complex. Of note, the analogous antagonist probe labeled a distinct region within the receptor amino terminus, confirming a key structural difference in active and inactive complexes.

Amino Acid Sequence↗
PubMed1991

Purification and N-terminal amino acid sequence of the Ah receptor from the C57BL/6J mouse.

The Ah receptor is a presumed member of the superfamily of steroid/thyroid hormone receptors, a trace soluble protein present in a wide variety of vertebrate species that mediates the biological effects of halogenated aromatic hydrocarbons. In this paper, we report the purification to homogeneity of this protein (from the liver of C57BL/6J mice) and its N-terminal amino acid sequence. Selective covalent labeling of the Ah receptor in hepatic cytosol with the photoaffinity ligands 2-azido-3-[125I]iodo-7,8-dibromodibenzo-p-dioxin simplified identification and quantitation of the receptor and permitted purification under denaturing conditions. Photoaffinity-labeled hepatic cytosol was applied to a phosphocellulose column at 80 mM NaCl, and the fraction enriched with the Ah receptor eluted with 225 mM NaCl. The eluate was diluted to 150 mM NaCl and applied to a DEAE-cellulose column, and the enriched fraction eluted with 300 mM. These two ion exchange chromatography steps usually gave approximately 100-fold enrichment and 40-50% recovery of Ah receptor. The dilute protein in the eluate was precipitated with n-propanol/trichloroacetic acid and solubilized in formic acid. The sample was then subjected to three successive rounds of high performance liquid chromatography on C4 reverse phase columns. The final, shallow-gradient chromatography was able to resolve the unlabeled 95-kDa receptor protein from the later eluting 125I-photoaffinity-labeled protein. The pooled high performance liquid chromatography fractions subjected to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels contained only the 95-kDa band upon staining with Coomassie blue R250 or silver. Using the above protocol, the Ah receptor was purified greater than 150,000-fold, to apparent homogeneity, with an overall yield of 3-5%. The N-terminal amino acid sequence of the purified peptide was determined to be ala/asp-ser-Arg-Lys-arg-Lys-Pro-Val-Gln-Lys-Thr-Val-Lys-Pro-Ile-Pro-Ala- Glu-Gly--Ile-Lys-ser-Asn-Pro-ser-Lys- (where the lowercase indicates a residue determined with less confidence).

Affinity Labels↗
PubMed1993

Modified taxols, 9. Synthesis and biological evaluation of 7-substituted photoaffinity analogues of taxol.

The 7-substituted taxol analogues 7, 19, 27, and 32 have been prepared as potential photoaffinity-labeled derivatives for studies of the nature of the binding site of taxol on polymerized tubulin. The analogue 32 has been prepared in both deuterium- and tritium-labeled versions. Tubulin-assembly studies were carried out with these compounds, and it was found that they showed some but not all of the properties of taxol. We conclude that these specific taxol analogues labeled at the 7 position are not ideal derivatives for photoaffinity labeling studies.

Affinity Labels↗
PubMed1996

Partial reversal of multidrug resistance in human breast cancer cells by an N-myristoylated protein kinase C-alpha pseudosubstrate peptide.

The predominant characteristics of multidrug resistant (MDR) cancer cells are broad spectrum resistance to chemotherapeutic agents and a pronounced defect in intracellular accumulation of the drugs, in association with overexpression of the drug efflux pump P-glycoprotein. Protein kinase C (PKC) phosphorylates the linker region of P-glycoprotein. Evidence has been presented that the isozyme PKC-alpha may contribute to the drug resistance phenotype of human breast cancer MCF7-MDR cells, PKC-alpha is markedly overexpressed in MCF7-MDR cells, and artificial overexpression of PKC-alpha in MCF7 constructs that overexpress P-glycoprotein significantly enhances the MDR phenotype of the cells in association with increased P-glycoprotein phosphorylation. Verapamil, cyclosporin A, and a number of other agents that compete with cytotoxic drugs for binding sites on P-glycoprotein can potently reverse MDR, but this is accompanied by severe toxicity in vivo. In this report, we demonstrate that an N-myristoylated peptide that contains a sequence corresponding to the pseudosubstrate region of PKC-alpha (P1) partially reverses multidrug resistance in MCF7-MDR cells by a novel mechanism that involves inhibition of PKC-alpha. P1 and two related PKC inhibitory N-myristoylated peptides restored intracellular accumulation of chemotherapeutic drugs in association with inhibition of the phosphorylation of three PKC-alpha substrates in MCF7-MDR cells: PKC-alpha, Raf-1 kinase, and P-glycoprotein. A fourth N-myristoylated peptide substrate analog of PKC, P7, did not affect drug accumulation in the MCF7-MDR cells and failed to inhibit the phosphorylation of the PKC-alpha substrates. The effects of P1 and verapamil on drug accumulation in MCF7-MDR cells were additive. P1 did not affect P-glycoprotein expression. MCF7-MDR cells were not cross-resistant to P1, which suggest that the peptide was not transported by P-glycoprotein. Furthermore, P1 was distinguished from MDR reversal agents such as verapamil and cyclosporin A by its inability to inhibit [3H]azidopine photoaffinity labeling of P-glycoprotein. P1 actually increased [3H] azidopine photoaffinity labeling of P-glycoprotein in MCF7-MDR cells, providing evidence that the effects of P1 on P-glycoprotein in MCF7-MDR cells are not restricted to inhibition of the phosphorylation of the pump. P1 may provide a basis for developing a new generation of MDR reversal agents that function by a novel mechanism that involves inhibition of PKC-alpha-catalyzed P-glycoprotein phosphorylation.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

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., Strömstedt, P.-E., Persson, B., Cederlund, E., Gustafsson, J.-A., and Jörnvall, 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.

Affinity Labels↗
PubMed1999

Cyclic adenosine 3':5'-monophosphate-dependent protein kinase on the external surface of LS-174T human colon carcinoma cells.

The analysis of purified plasma membranes and the surface of intact cells revealed the presence of cyclic adenosine 3':5'-monophosphate-(cAMP) dependent protein kinase (PKA) on the external surface of LS-174T human colon carcinoma cells. Photoaffinity labeling of intact cells at confluence with 8-azido-[32P]cAMP identified the cAMP-binding proteins on the surface. Immunoprecipitation identified the photoaffinity-labeled cAMP-binding proteins as the RIIalpha regulatory subunit of PKA. During the logarithmic stage of growth, both the RIalpha and RIIalpha subunits of PKA were localized on the cell surface. Intact LS-174T cells catalyzed the phosphorylation of Kemptide in a cAMP-dependent manner; upon substitution of cAMP in the medium with 8-chloroadenosine, which did not compete with cAMP for the binding on intact cells, the ecto-PKA was no longer activated. The specific inhibitory protein for PKA, PKI, abolished the stimulation of phosphorylation by cAMP. Forskolin, which elevates intracellular levels of cAMP, activated ecto-PKA. Moreover, probenecid, which blocks the export of cAMP, inhibited the forskolin-mediated activation of ecto-PKA. These results demonstrate that LS-174T colon carcinoma cells possess an ecto-PKA on the external surface. This ecto-PKA is similar, if not identical, to the soluble intracellular PKA.

Azides↗
PubMed2000

A high-affinity fluorenone-based beta 2-adrenergic receptor antagonist with a photoactivatable pharmacophore.

To develop molecules capable of directly probing the catechol binding region of the beta(2)-adrenergic receptor (beta(2)AR), novel benzophenone- and fluorenone-based beta(2)AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone+ ++ (iodoaminoflisopolol, IAmF), showed very high affinity for the beta(2)AR, inhibiting [(125)I]ICYP binding with an apparent K(i) of approximately 1 x 10(-)(9) M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon-carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous beta(2)AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta(2)AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the beta(2)AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([(125)I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [(125)I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta(2)AR in membranes (protectable by 10 microM alprenolol) was demonstrated. [(125)I]IAmF represents a new class of beta(2)AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the beta(2)AR ligand binding pocket.

Adenylyl Cyclase Inhibitors↗