Testicular GnRH receptors: photoaffinity labeling and fluorescence distribution studies

Specific GnRH receptor proteins of purified rat Leydig cells and membrane preparations were identified using an 125I-labeled bioactive photoaffinity derivative of GnRH. Sodium dodecyl sulfate polyacrylamide gel electrophoresis resulted in the identification of two specific components with apparent molecular weights of 60,000 and 54,000 daltons. Fluorescent visualization of GnRH receptors in these cells, utilizing a bioactive rhodamine derivative of the hormone, indicated that the fluorescently labeled receptors were initially distributed uniformly on the cell surface and then formed clusters which subsequently internalized (at 37 degrees C) into endocytic vesicles. These processes were dependent on specific binding sites for the rhodamine-labeled peptide on Leydig cells. These findings indicate further characterization of the testicular GnRH receptors and may have important implications towards the understanding of the molecular events involved in the action of the hormone in the testis.     

Fluorescent and photoaffinity labeling probes for retinoic acid receptors.

A fluorescent probe for retinoid receptors (RARs) was designed and prepared. The probe consists of a retinoid moiety and a dansyl moiety, i.e., 2-[3-(5-dimethylaminonaphthalene-1-sulfonyl)- aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2- naphthalenyl)carboxamido]benzoic acid: DAM-3. DAM-3 specifically bound RARs. Additionally, a photoreactive RAR fluorescent probe was designed and prepared, i.e., 2-[3-(5-azidonaphthalene- 1-sulfonyl)aminopropyl-1-oxy]-4-[(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)carboxamido]benzoic acid (ADAM-3). ADAM-3 irreversibly and specifically bound RARs using ultraviolet irradiation.     

Insulin receptors are bivalent as demonstrated by photoaffinity labeling.

Insulin receptors in human placental membranes were photoaffinity-labeled with a radioactive human insulin-like growth factor I (hIGF-I) photoprobe N epsilon B28-monoazidobenzoyl 125I-hIGF-I either alone or together with a non-radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl insulin. Precipitation of the solubilized receptors with anti-insulin antibody showed that receptors labeled with the radioactive hIGF-I photoprobe were detected in the immunoprecipitate only when photolabeling was carried out in the presence of the non-radioactive insulin photoprobe. Comparable results were obtained in converse experiments using a radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl 125I-insulin, a non-radioactive hIGF-I photoprobe N epsilon B28-monoazidobenzoyl hIGF-I, and an antibody to hIGF-I. The amount of radioactive receptors precipitated by either the anti-insulin antibody or the anti-hIGHF-I antibody was close to the expected amount. These observations demonstrate that the insulin receptor is bivalent being capable of binding two molecules of ligand.     

Solubilization and photoaffinity labeling of renal membrane cyclic AMP receptors.

Renal cortical plasma membranes were solubilized with sodium deoxycholate. The membrane-bound cyclic AMP receptors retained biologic activity in the detergent-dispersed state exhibiting the properties of high affinity for cyclic AMP, saturability and specificity. Half-maximal binding of cycle [3H]-AMP to these receptors was found to occur at 0.06 muM and 1.5 pmol of cyclic [3H]AMP was bound per mg membrane protein at saturation (0.5 muM cyclic [3H]AMP). Sodium deoxycholate-solubilized membrane proteins were chromatographed on Biogel A-5m. Cyclic [3H]AMP receptors eluted in the internal volume at positions equivalent to molecular sizes of 50 000 and 20 000 daltons and in the void volume at molecular size greater than 450 000. After photoaffinity labeling the renal membrane receptors with cyclic [3H]AMP, we found peaks of tritium radioactivity which eluted at similar molecular size positions on this Bogel A-5m column. Further treatment of photoaffinity labeled membranes with sodium dodecyl sulfate, mercaptoethanol and urea, followed by polyacrylamide gel electrophoresis, showed bands of tritium-labeled receptor protein with relative mobilities corresponding to molecular sizes of 26 000 and 21 000 daltons. This study shows that porcine renal cortical membranes contain at least two molecular species of cyclic AMP receptors which may be associated with regulation of the membrane-bound cyclic AMP-dependent protein kinase.     

Diazirine based photoaffinity labeling

Diazirines are among the smallest photoreactive groups that form a reactive carbene upon light irradiation. This feature has been widely utilized in photoaffinity labeling to study ligand-receptor, ligand-enzyme and protein-protein interactions, and in the isolation and identification of unknown proteins. This review summarizes recent advances in the use of diazirines in photoaffinity labeling.     

Identification of thyroid hormone receptors in rat liver nuclei by photoaffinity labeling with L-thyroxine and triiodo-L-thyronine

Photoaffinity labeling of rat liver nuclear extract with underivatized thyroid hormones was performed after incubation with 1 nM [3',5'-125I]thyroxine ([125I]T4) or [3'-125I]triiodothyronine [( 125I]T3) by irradiation with light above 300 nm. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the covalently photolabeled nuclear extract revealed four distinct hormone binding proteins of molecular masses 96, 56, 45, and 35 kilodaltons (kDa), respectively. Distribution of the hormone among these proteins was similar for T4 and T3. The 56- and 45-kDa proteins were the most prominently labeled. The specificity of the photoattachment of thyroid hormones to these nuclear proteins was verified by the irradiation of eight randomly chosen proteins and two proteins known to have thyroid hormone binding sites, human thyroxine binding globulin and bovine serum albumin. Only the latter two were photolabeled with [125I]T4. Competition studies performed by incubating nuclear extracts with [125I]T4 or [125I]T3 in the presence of increasing amounts of the corresponding unlabeled hormone (10-, 100-, and 1000-fold molar excess) demonstrated that (1) photoattachment of labeled T3 or T4 to the 56- and 45-kDa proteins was inhibited by 67-78% and 73-85%, respectively, after incubation with a 1000-fold molar excess of unlabeled hormone, (2) in the presence of lower molar excesses of the corresponding competitor (10- and 100-fold), photoattachment of labeled T3 or T4 to the 56- and 45-kDa receptors was gradually inhibited to a similar extent on both proteins, and (3) the 35- and 96-kDa proteins, although having thyroid hormone binding sites, display lower binding activities since the inhibition of photoattachment of labeled T3 or T4 by a 1000-fold molar excess of unlabeled hormone did not exceed 30-42% and 26-49%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and characterization of adrenal and uterine angiotensin II receptors after photoaffinity labeling

The physical characteristic of the receptors for angiotensin II in dog adrenal cortex and uterus were determined after affinity labeling. 125I-nitro-5-azido-benzoyl-angiotensin II, a photosensitive angiotensin II analogue which retained aldosterone-stimulating activity, was used to couple the octapeptide specifically and irreversibly to its membrane receptors. After solubilization with Triton X-100, the covalent hormone . receptor complex was analyzed by gel filtration and sucrose density gradient centrifugation. Two radioactive species were consistently observed, with calculated Mr values of 126,000 +/- 10,000 and 64,500 +/- 11,000. the elution profiles of solubilized adrenal and uterine particles were almost identical. When the solubilized complexes were subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, a single radioactive band was detected upon autoradiography, with Mr - 65,000 +/- 6,000 for adrenal cortex and 68,000 +/- 7,000 for myometrium. These results indicate that the receptors for angiotensin II in adrenal cortex and uterus are composed of two subunits of similar molecular weight, and that the common functional properties of the receptors from both tissues are probably related to their similar physicochemical characteristics.     

Persistent activation of steroidogenesis in adrenocortical cells by photoaffinity labeling of corticotropin receptors

Photolysis of rat adrenocortical cells in the presence of the photoreactive derivative [(2-nitro-5-azidophenylsulfenyl)Trp9]-adrenocorticotropic hormone (2,5-NAPS-ACTH) at 24 degrees C resulted in persistent activation of corticosterone production. The basal rate of steroidogenesis became maximal when photolysis was performed at 24 degrees C but remained the same as that of control cells when irradiation was performed at 0 degrees C. No increase in basal rate was observed with dark controls or cells photolyzed with [(2,4-dinitrophenylsulfenyl)Trp9]ACTH, a photoresistant analog of the hormone. Prephotolyzed 2,5-NAPS-ACTH failed to induce persistent activation. Both ACTH and 2,4-(dinitrophenylsulfenyl)Trp9-ACTH blocked the photo-induced activation of steroidogenesis elicited by 2,5-NAPS-ACTH. Under photolysis conditions which caused the basal rate of steroidogenesis to become maximal, a 3-fold increase in the basal rate of cAMP formation was observed.     

p-Toluenesulfonyldiazoacetates: Reagents for photoaffinity labeling

p-Toluenesulfonyldiazoacetyl chloride and p-nitrophenyl p-toluenesulfonyldiazoacetate have been prepared and offer potential advantages as reagents for photoaffinity labeling. (i) The extinction coefficient for the sulfonyldiazo compounds at 370 nm is about 10 times that for the long wavelength absorption of other diazoesters; this absorption permits reasonably rapid photolysis in the presence of compounds that are destroyed by short wavelength uv radiation. (ii) The two derivatives named above are stable thermally; furthermore, since sulfonyldiazoesters are stable to acid and to weak base, photoaffinity labeling can be conducted over a wide range of pH. (iii) Photolysis of ordinary (i.e., oxygen) esters of sulfonyldiazo compounds in methanol or cyclohexane leads to insertion into the solvent to the exclusion of Wolff rearrangement; photolysis of thioesters at 350 nm in methanol gives about 25% insertion into solvent, accompanied by about 75% Wolff rearrangement; in contrast, photolysis of most thioesters of diazo derivatives leads exclusively to Wolff rearrangement     

Dextrorphan: an antagonist for phencyclidine receptors

Radio-binding assay, bioassay and HPLC detection were used to observe the antagonistic effects of dextrorphan on PCP's actions. Dextrorphan displayed high affinity to PCP receptor in the rabbit mesenteric blood vessels. It had weak PCP-like bioactivity, but could antagonize PCP's action dose-dependently in vitro study with the rabbit ear artery preparation and shifted the dose-response curve of PCP to the right. After PCP administration, the content of norepinephrine in the vascular bath medium was increased, which was reversed by dextrorphan. Thus suggests that dextrorphan is an antagonist with very mild agonistic action for PCP receptors.     

Effects of pH and temperature on photoaffinity labeling of Family B G protein-coupled receptors

The efficiency of covalent labeling of a receptor by a photolabile analogue of its natural ligand is dependent on the spatial approximation of the probe and its target. Systematic application of intrinsic photoaffinity labeling to the secretin receptor, a prototypic Family B G protein-coupled receptor, demonstrated reduced efficiency of labeling for amino-terminal and mid-region sites of labeling relative to carboxyl-terminal sites. Reduction of pH from 7.4 to 5.5 and reduction of temperature from 25 °C to 4 °C improved the efficiency of covalent labeling of the receptor with these probes. This correlated with sites of labeling at the interface between the receptor amino terminus and the receptor core, a region containing histidine residues that have their ionization affected in this pH range. Application to the calcitonin receptor, another Family B G protein-coupled receptor, yielded analogous results. These results support the consistent mode of docking peptide ligands to this group of receptors.     

Direct photoaffinity labeling of tubulin with guanosine 5'-triphosphate

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.     

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human platelet alpha 2-adrenergic receptors: labeling with 3H-yohimbine, a selective antagonist ligand

³H-Yohimbine, a potent and selective pharmacological antagonist of α₂-adrenergic receptors, labeled human platelet membrane α₂-receptors with high affinity. Binding was rapid and reversible at 25°C. Both saturation and kinetic experiments indicated a single order of binding sites, with an equilibrium K_D value of 1.0–1.5 nM. Low Mg²⁺ concentrations increased the K_D for ³H-yohimbine without altering the B_max. The ³H-yohimbine site exhibited α₂-receptor specificity: (?)-norepinephrine and (?)-isoproterenol were 4.8 and 330 times less potent than (?)-epinephrine; (?)-catecholamines were 17–35 times more potent than corresponding (+)-catecholamines; the selective α₁-antagonist prazosin was 340 times less potent than yohimbine. Catecholamine agonists exhibited shallow curves in inhibiting ³H-yohimbine binding, with pseudo-Hill coefficients (n_H) of less than 1.0, whereas the n_H of antagonists was 1.0. No specific binding of ³H-prazosin to platelet membranes was observed, indicating the absence of α₁-receptors. ³H-Yohimbine labeled fewer platelet sites than did ³H-dihydroergocryptine under identical conditions (80 vs 130 receptors/ cell), and may be a more specific and useful antagonist probe of platelet α₂-receptors than ³H-dihydroergocryptine.     

Active cytokinins: photoaffinity labeling agents to detect binding

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N⁶-benzyladenines, -N⁶-(Δ²-isopentenyl)adenines, and -zeatins, and N⁶-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10⁻⁵ micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10⁻⁶ micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10⁻⁵ micromolar, representing a sensitivity rarely attained.     

Direct photoaffinity labeling of gizzard myosin with vanadate-trapped adenosine diphosphate

The active-site topology of smooth muscle myosin has been investigated by direct photoaffinity-labeling studies with [3H]ADP. Addition of vanadate (Vi) and Co2+ enabled [3H]ADP to be stably trapped at the active site (t1/2 greater than 5 days at 0 degrees C). The extraordinary stability of the myosin.Co2+.[3H]ADP.Vi complex allowed it to be purified free of excess [3H]ADP before irradiation began and ensured that only active-site residues became labeled. Following UV irradiation, approximately 10% of the trapped [3H]ADP became covalently attached at the active site. All of the [3H]ADP incorporated into the 200-kDa heavy chain, confirming earlier results using untrapped [alpha-32P]ATP [Maruta, H., & Korn, E. (1981) J. Biol. Chem. 256, 499-502]. After extensive trypsin digestion of labeled subfragment 1, HPLC separation methods combined with alkaline phosphatase treatment allowed two labeled peptides to be isolated. Sequence analysis of both labeled peptides indicated that Glu-185 was the labeled residue. Since Glu-185 has been previously identified as a residue at the active site of smooth myosin using [3H]UDP as a photolabel [Garabedian, T. E., & Yount, R. G. (1990) J. Biol. Chem. 265, 22547-22553], these results provide further evidence that Glu-185, located immediately adjacent to the glycine-rich loop, is located in the purine binding pocket of the active site of smooth muscle myosin.     

In situ photoaffinity labeling of proteasome with photoactive adriamycin analogue

An intracellular adriamycin (ADM)-binding protein purified from the cytosol of L1210 mouse lymphocytic leukemia cells had a molecular weight of 700-1500 kDa and hydrolyzed Suc-LLVY-MCA. When L1210 cells were incubated with a photoactive ADM analogue, N-(p-azidobenzoyl)-adriamycin (NAB-ADM), most of the NAB-ADM was found to localize in the nuclei. In situ photoaffinity labeling of L1210 cells with NAB-ADM resulted in low protease activity in the cytosol and nuclear extracts and the cells showed selective photoincorporation of NAB-ADM into the proteasome. These results suggest that the proteasome is a translocator of ADM from the cytoplasm to the nucleus and might therefore become a new candidate for cancer chemotherapy.     

Fluorescent and photoaffinity labeling derivatives of rhizoxin

A fluorescent probe (D-RZX) and a photoreactive fluorescent probe (AD-RZX) for studying the rhizoxin binding site on tubulin were prepared by the derivatization of rhizoxin (RZX). D-RZX consists of a rhizoxin moiety and a dansyl moiety. AD-RZX has a 5-azidonaphthalene-1-sulfonyl moiety instead of the dansyl moiety of D-RZX. Both D-RZX and AD-RZX bound tubulin in a mutually competitive manner with rhizoxin, indicating their binding to the rhizoxin site on tubulin. AD-RZX bound the rhizoxin site covalently after UV-irradiation, thus showing its usefulness as a photo-affinity probe for labeling of the rhizoxin site.     

Direct photoaffinity labeling of leukotriene binding sites

Due to their conjugated double bonds the leukotrienes themselves are photolabile compounds and may therefore be used directly for photoaffinity labeling of leukotriene binding sites. Cryofixation eliminates unspecific labeling taking place in solution by photoisomers and photodegradation products of leukotrienes. After fixation of receptor ligand interactions by shock-freezing of the samples, irradiation-induced highly reactive excited states and/or intermediates can form covalent bonds with the respective binding site in the frozen state. After cryofixation of a solution of albumin incubated with [3H8]leukotriene E4, irradiation at 300 nm resulted in time-dependent incorporation of radioactivity into the protein. Photoaffinity labeling of rat as well as of human blood serum with [3H8]leukotriene E4 after cryofixation revealed that only one polypeptide with an Mr of 67,000 was labeled. This polypeptide was identified as albumin. Photoaffinity labeling of rat liver membrane subfractions enriched with sinusoidal membranes resulted in the labeling of a polypeptide with an apparent Mr of 48,000, whereas no polypeptide was predominantly labeled in the subfraction enriched with canalicular membranes. Photoaffinity labeling of isolated hepatocytes disclosed different leukotriene E4 binding polypeptides. In the particulate fraction of hepatocytes a polypeptide with an apparent Mr of 48,000 was labeled predominantly, whereas in the soluble fraction several polypeptides were labeled to a similar extent. One of these, with an apparent Mr of 25,000, was identified as subunit 1 of glutathione transferases by immunoprecipitation. The method of direct photoaffinity labeling in the frozen state after cryofixation using leukotrienes as photoactivatable compounds, as exemplified by leukotriene E4, may be most useful for the identification and characterization of various leukotriene binding sites, including receptors, leukotriene-metabolizing enzymes, and transport systems.