The major site of photoaffinity labeling of the gamma-aminobutyric acid type A receptor by [3H]flunitrazepam is histidine 102 of the alpha subunit

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).     

Structural studies of the H+/oligopeptide transport system from rabbit small intestine

A 127-kDa protein was identified as a component of the H+/oligopeptide transport system in brush-border membrane vesicles from rabbit small intestine by photoaffinity labeling with [3H]cephalexin and further photoreactive beta-lactam antibiotics and dipeptides. Reconstitution of stereospecific transport activity revealed the involvement of the 127-kDa protein in H+-dependent transport of oligopeptides and orally active alpha-amino-beta-lactam antibiotics (Kramer et al., Eur. J. Biochem. 204 (1992) 923-930). H+-Dependent transport activity was found in all segments of the small intestine concomitantly with the specific labeling of the 127-kDa protein. By enzymatic deglycosylation, fragments of Mr 116 and 95 kDa were obtained from the 127-kDa protein with endoglucosidase F and N-glycanase, whereas with endoglucosidase H, a fragment of Mr 116 kDa was formed. These findings indicate that the photolabeled 127-kDa protein is a microheterogenous glycoprotein. Surprisingly, it was found that the solubilized and purified 127-kDa protein showed enzymatic sucrase and isomaltase activity. Inhibition of the glucosidase activities with the glucosidase inhibitor HOE 120 influenced neither H+/oligopeptide transport nor photoaffinity labeling of the 127-kDa protein. With polyclonal antibodies raised against the purified 127-kDa protein, a coprecipitation of sucrase activity and the photolabeled 127-kDa beta-lactam antibiotic binding protein occurred. Target size analysis revealed a functional molecular mass of 165+/-17 kDa for photoaffinity labeling of the 127-kDa protein, suggesting a homo- or heterodimeric functional structure of the 127-kDa protein in the brush-border membrane. These findings indicate that the H+/oligopeptide binding protein of Mr 127000 is closely associated with the sucrase/isomaltase complex in the enterocyte brush-border membrane.     

Differential T cell receptor photoaffinity labeling among H-2Kd restricted cytotoxic T lymphocyte clones specific for a photoreactive peptide derivative. Labeling of the alpha-chain correlates with J alpha segment usage

Using a direct binding assay based on photoaffinity labeling, we studied the interaction of T cell receptor (TCR) with a Kd-bound photoreactive peptide derivative on living cells. The Kd-restricted Plasmodium berghei circumsporozoite (PbCS) peptide 253-260 (YIPSAEKI) was reacted NH2-terminally with biotin and at the TCR contact residue Lys259 with photoreactive iodo, 4-azido salicylic acid (IASA) to make biotin-YIPSAEK(IASA)I. Cytotoxic T lymphocyte (CTL) clones derived from mice immunized with this derivative recognized this conjugate, but not a related one lacking the IASA group nor the parental PbCS peptide. The clones were Kd restricted. Recognition experiments with variant conjugates, lacking substituents from IASA, revealed a diverse fine specificity pattern and indicated that this group interacted directly with the TCR. The TCR of four clones could be photoaffinity labeled by biotin-YIPSAEK(125IASA)I. This labeling was dependent on the conjugates binding to the Kd molecule and was selective for the TCR alpha (2 clones) or beta chain (1 clone), or was common for both chains (1 clone). TCR sequence analysis showed a preferential usage of J alpha TA28 containing alpha chains that were paired with V beta 1 expressing beta chains. The TCR that were photoaffinity labeled at the alpha chain expressed these J alpha and V beta segments. The tryptophan encoded by the J alpha TA28 segment is rarely found in other J alpha segments. Moreover, we show that the IASA group interacts preferentially with tryptophan in aqueous solution. We thus propose that for these CTL clones, labeling of the alpha chain occurs via the J alpha-encoded tryptophan residue.     

Specific photoaffinity labeling of Tyr-49 on the light chain in the steroid-combining site of a mouse monoclonal anti-estradiol antibody using two epimeric 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amidoestradiol photoreagents

A mouse monoclonal anti-7-(O-carboxymethyl)oximinoestradiol antibody was photoaffinity labeled with two cross-reactive 6alpha- and 6beta-(5-azido-2-nitrobenzoyl)amido[17alpha-3H]estradiol photoreagents (6alpha- and 6beta-ANBA-[17alpha-3H]estradiol). Covalently bound radioactivity was found exclusively on the light chain. The maximal level of specific incorporation was 0.18 mol of label per mole of antibody for both photoreagents. In both cases, tryptic digestion of the photolabeled light chain, immunopurification with the immobilized antibody, reverse-phase liquid chromatography, and Edman degradation showed the presence of radioactive peptide GLM-([3H]X)-HGNTLEDGIPSR derived from peptide 46-61 of the light chain sequence (determined from cDNA) in which the unidentified amino acid corresponding to X is a Tyr residue. Two other radioactive peptides were also isolated, one corresponding probably to the methionine sulfoxide derivative of the peptide 46-61 photolabeled with the 6beta-reagent and the other to the N-terminal tetrapeptide 46-49 of the peptide 46-61 photolabeled with the 6alpha-reagent. In all cases, the main peak of radioactivity was released at the fourth Edman cycle, thus suggesting that the same Tyr-49 residue on the light chain was photolabeled. This residue is contiguous to the N-terminal amino acid of the second hypervariable complementary determining region 50-56 of light chain. Covalent labeling was confirmed by mass spectrometry of photolabeled peptides which showed molecular ion values corresponding to the addition of the photoactive 6alpha- or 6beta-ANBA-estradiol nitrene derivatives to the peptide.     

Dopamine transporter ligand binding domains. Structural and functional properties revealed by limited proteolysis

Dopamine transporters (DATs) are members of the Na+- and Cl--dependent neurotransmitter and amino acid transporter family predicted by hydrophobicity analysis to have 12 transmembrane-spanning helices. The structure of DAT was studied using the photoaffinity compounds [125I]1-[2-(diphenylmethoxy)-ethyl]-4-[2-(4-azido-3-iodophenyl) ethyl] piperazine ([125I]DEEP), a 1-(2-diphenylmethoxy)-ethyl-4-(3-phenyl propyl)piperazine (GBR analog), and [125I]-3beta-(p-chlorophenyl)tropane-2beta-carboxylic acid, 4'-azido-3'-iodophenylethyl ester ([125I]RTI 82), a cocaine analog, which had been shown in a previous study to become incorporated into different regions of the DAT primary sequence. The proximity of the photolabeled binding sites to integral membrane structures was investigated by subjecting photolabeled membrane suspensions to limited proteolysis with trypsin and separately analyzing the resulting membranes and supernatants for the presence of photolabeled DAT fragments. Trypsin treatment of [125I] DEEP-labeled membranes generated labeled 45- and 14-kDa DAT fragments that immunoprecipitated with an epitope-specific antiserum generated against amino acids 42-59 near the first putative transmembrane domain, whereas [125I]RTI 82 was found in 32- and 16-kDa tryptic fragments that precipitated with an antiserum directed against a sequence near transmembrane domain 4 (amino acids 225-238). All of the photolabeled fragments were recovered in the protease-treated membranes, indicating that they possess integral membrane structures that prevent their release from the membrane as soluble forms. The size of the two smallest fragments in conjunction with their retention in the membrane suggests that incorporation of the photoaffinity ligands occurs in or near membrane spanning regions and delineates the maximum possible distance between the transmembrane structures, incorporated photolabel, and antibody epitopes. Carbohydrate analysis of the fragments identified sialic acids and N-linked oligosaccharides exclusively on the 45-kDa [125I]DEEP-labeled fragment, which, based on size, would be expected to contain four consensus glycosylation sites between putative transmembrane domains 3 and 4. Photoaffinity labeling after trypsin treatment of membranes showed that the larger but not the smaller fragments retain binding capacity, as the 45- and 32-kDa fragments were capable of becoming photolabeled. Binding of photoaffinity ligands at these fragments was displaced with the same pharmacology as that of intact DATs. These results verify numerous aspects of DAT structure and topology heretofore only predicted from theoretical considerations and extend our knowledge of DAT structure-function properties.     

Topological photoaffinity labeling of the rabbit ileal Na+/bile-salt-cotransport system

For the investigation of the topology of the rabbit ileal Na+/bile-salt-cotransport system, composed of a 93-kDa integral membrane protein and a peripheral 14-kDa bile-acid-binding protein (ILBP), we have synthesized photolabile dimeric bile-salt-transport inhibitors (photoblockers), G1-X-G2, where two bile acid moieties (G1 and G2) are tethered together via a spacer, X, and where one of the two bile acid moieties carries a photoactivatable group. These photoblockers specifically interact with the ileal Na+/bile-salt-cotransport system as demonstrated by a concentration-dependent inhibition of [3H]cholyltaurine uptake by rabbit ileal brush-border membrane vesicles and by inhibition of photolabeling of the 93-kDa and 14-kDa bile-salt-binding proteins by 7,7-azo and 3,3-azo derivatives of cholyltaurine. Ileal bile-salt uptake was specifically inhibited by the photoblockers, which were not taken up themselves by the small intestine as demonstrated by in vivo ileal perfusion. Dependent on the photoblocker used several polypeptides in the molecular-mass range of 14-130 kDa were labeled. The cytoplasmically attached 14-kDa ILBP was significantly labeled only by inhibitors that are photoactivatable in bile acid moiety G1, suggesting that during binding and translocation of a bile-salt molecule by the ileal bile-salt-transport system the steroid nucleus gets access to the cytoplasmic site of the ileal brush-border membrane first. Photoaffinity labeling in the frozen state with the transportable 3,3-azo and 7,7-azo derivatives of cholyltaurine revealed a time-dependent increase in the extent of labeling of the 14-kDa and 93-kDa proteins, suggesting a labeling of these proteins from the cytoplasmic site of the ileal brush-border membrane. By photoaffinity labeling in the frozen state with the various photoblockers time-dependent changes in the extent of photoaffinity labeling of bile-salt-binding proteins were observed, demonstrating the possibility of topological analysis of the rabbit ileal Na+/bile-salt-cotransport system.     

Characterization of AT4 receptor from bovine aortic endothelium with photosensitive analogues of angiotensin IV

Newly developed photosensitive analogues of AngIV were used to characterize the AT4 receptor of bovine aortic endothelial cells. The photoactivatable AngIV analogues [N3-Phe6]AngIV and [Bpa6]AngIV displayed high affinities for AT4 receptor, with IC50's of 3.7 +/- 0.3 and 19.1 +/- 3.5 nM, respectively. The radioiodinated ligands showed a good efficiency of photoaffinity labeling demonstrated by high proportions (60-75%) of acid-resistant binding. Covalently labeled receptor was solubilized under reducing or nonreducing conditions and subjected to SDS-PAGE. Under nonreducing conditions, autoradiographies revealed a major band of Mr 186 +/- 2 kDa and a minor band of Mr 241 +/- 6 kDa. The labeling of these bands was completely abolished in the presence of 10 microM AngIV. Under reducing conditions, only the low Mr 186 kDa band was revealed. After endoglycosidase digestion with an enzyme that cleaves N-linked saccharides, the Mr of the denatured AT4 receptor was decreased by 31% to a value of 129 +/- 10 kDa. Kinetic studies revealed a stepwise process of AT4 receptor deglycosylation by endoglycosidase F, suggesting at least two different sites of N-linked saccharides. Mild trypsin treatment of photolabeled endothelial cell membranes released a large fragment of Mr 177 +/- 3 kDa which accounts for about 95% of the whole receptor molecular mass. These results demonstrate that [N3-Phe6]AngIV and [Bpa6]AngIV are very efficient tools for selective photoaffinity labeling of AT4 receptor. We have shown that AT4 receptor is a 186 kDa integral membrane glycoprotein with a very large extracellular domain. These properties are consistent with those of a growth factor or cytokine receptor.     

Differential roles of T cell receptor alpha and beta chains in ligand binding among H-2Kd-restricted cytolytic T lymphocyte clones specific for a photoreactive Plasmodium berghei circumsporozoite peptide derivative

To study the interaction of T cell receptor with its ligand, a complex of a major histocompatibility complex molecule and a peptide, we derived H-2Kd-restricted cytolytic T lymphocyte clones from mice immunized with a Plasmodium berghei circumsporozoite peptide (PbCS) 252-260 (SYIPSAEKI) derivative containing photoreactive Nepsilon-[4-azidobenzoyl] lysine in place of Pro-255. This residue and Lys-259 were essential parts of the epitope recognized by these clones. Most of the clones expressed BV1S1A1 encoded beta chains along with specific complementary determining region (CDR) 3beta regions but diverse alpha chain sequences. Surprisingly, all T cell receptors were preferentially photoaffinity labeled on the alpha chain. For a representative T cell receptor, the photoaffinity labeled site was located in the Valpha C-strand. Computer modeling suggested the presence of a hydrophobic pocket, which is formed by parts of the Valpha/Jalpha C-, F-, and G-strands and adjacent CDR3alpha residues and structured to be able to avidly bind the photoreactive ligand side chain. We previously found that a T cell receptor specific for a PbCS peptide derivative containing this photoreactive side chain in position 259 similarly used a hydrophobic pocket located between the junctional CDR3 loops. We propose that this nonpolar domain in these locations allow T cell receptors to avidly and specifically bind epitopes containing non-peptidic side chains.     

Identification of the benzomorphan opiate binding site on the catalytic subunit of acetylcholinesterase

The interaction of a benzomorphan opiate with the active site of the catalytic subunit of acetylcholinesterase was studied using photoaffinity labeling. UV irradiation of (-)-N-[3H]allylnormetazocine bound to Torpedo acetylcholinesterase resulted in covalent incorporation of 60-70% of the bound ligand. The labeled catalytic subunit was subjected to chemical cleavage with cyanogen bromide and proteolytic degradation with trypsin, chymotrypsin, and staphylococcal V8 protease. The resulting peptide fragments were purified by high performance liquid chromatography and sequenced in the gas phase. The label was not stable under the conditions of the sequencing, but a peptide fragment consisting of Gln74 to Glu82 was reproducibly labeled. These amino acids are located at the rim of a gorge leading to the active site of the enzyme. Molecular modeling studies then demonstrated that these residues can be placed within van der Waals contact of the (-)-N-[3H]allylnormetazocine molecule while it is bound to the active site of the enzyme.     

Identification of benz(othi)azepine-binding regions within L-type calcium channel alpha1 subunits

To identify the binding domain for diltiazem-like Ca2+ antagonists on L-type Ca2+ channel alpha1 subunits we synthesized the benzazepine [3H]benziazem as a novel photoaffinity probe. [3H]Benziazem reversibly labeled the benzothiazepine (BTZ)-binding domain of partially purified skeletal muscle Ca2+ channels with high affinity (Kd = 12 nM) and photoincorporated into its binding domain with high yield (>66%). Antibody mapping of proteolytic labeled fragments revealed specific labeling of regions associated with transmembrane segments S6 in repeats III and IV. More than 50% of the labeling was found in the tryptic fragment alanine 1023-lysine 1077 containing IIIS6 together with extracellular and intracellular amino acid residues. The remaining labeling was identified in a second site comprising segment S6 in repeat IV and adjacent residues. Unlike for dihydropyridines, no labeling was observed in the connecting IIIS5-IIIS6 linker. The [3H]benziazem photolabeled regions must be in close contact to the drug molecule when bound to the channel. We propose that the determinants for high affinity BTZ binding are located within or in close proximity to segments IIIS6 and/or IVS6. Therefore the binding domain for BTZs, like for the other main classes of Ca2+ antagonists, must be located in close proximity to pore-forming regions of the channel.     

Quantitative approach for detection and characterization of formyl peptide receptor in solution using a photoaffinity ligand

In this paper we describe an assay method for the rapid detection and quantitation of human neutrophil FMLP (N-formyl-methionyl-leucyl-phenylalanine) receptor molecules. Although different assay methods are available to detect the receptor, none are rapid and at the same time allow quantitative detection of the binding affinity of the receptor molecule in solution. In our modified method, following a binding reaction using photoaffinity ligand, the amount of labeled ligand bound to the receptor is separated from the unbound one, thereby determining the binding affinity of the receptor protein. This simple procedure not only makes it possible to detect the recombinant FMLP receptor protein very rapidly, but also provides quantitative assessment of binding. This technique therefore allows a partial characterization (identification, determination of size and assessment of binding affinity) of receptor molecule in solution in less than 24 hours.     

Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fractions from the Q-Sepharose, heparin-Sepharose, and WGA-agarose also indicated only the single radiolabeled band at 80 kDa. Intensity of photolabeling in each of the fractions examined coincided with enzyme activity. The photolabeling of this 80-kDa protein was saturable with the photoprobe and could be inhibited by the addition of UDP-GlcA prior to the addition of the photoprobe. Thus, the photolabeling with [beta-32P]5-azido-UDP-GlcA has identified the GlcA transferase II as an 80-kDa protein. The purified enzyme was capable of transferring good amounts of GlcA residues to chondroitin-derived pentasaccharide with negligible transfer to pentasaccharides derived from hyaluronan or heparan.     

Molecular weight determination of the hepatic vasopressin receptor with a high-affinity photoprobe

We report here a study of photoaffinity labeling of the V1a-vasopressin receptor with high-affinity, V1-specific radioiodinated antagonist ligands: one containing an azidophenylalanine residue ([beta,beta-dimethyl-beta-mercaptopropionyl(1), p-azido-Phe2,Val4,Lys8,D-Tyr9] vasopressin), two others containing nitrophenylalanine, and one, highly similar but without a photosensitive function, as control. All analogues competed in the dark for the same binding site with vasopressin. Long-wavelength UV irradiation of rat liver membranes incubated in presence of the radio-iodinated azido photolabel produced a specifically labeled protein band at 53 kDa in SDS-PAGE. Identical experiments with the nitrophenylalanyl peptides produced only non-specific labeling and control experiments with the non-photosensitive analogue produced no labeling at all. Chemical crosslinking of 3H-VP to the same membrane preparation produced a result identical to that of the azido photolabel, confirming the receptor nature of the labeled protein. Deglycosylation of the labeled receptor with endoglycosidase F reduced the observed molecular weight of 53 kDa to 43 kDa. The molecular parameters reported herein of the presumed hepatic vasopressin receptor confirm the values deduced from the molecular cloning of the rat V1a receptor.     

Demonstration of a direct interaction between residue 22 in the carboxyl-terminal half of secretin and the amino-terminal tail of the secretin receptor using photoaffinity labeling

An understanding of the molecular basis of hormonal activation of receptors provides important insights for drug design. Toward this end, intrinsic photoaffinity labeling is a powerful tool to directly identify the ligand-binding domain. We have developed a new radioiodinatable agonist ligand of the secretin receptor that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) into the position of Leu22 and have utilized this to identify the adjacent receptor domain. The rat [Tyr10,Bpa22]secretin-27 probe was a fully efficacious agonist, with a potency to stimulate cAMP accumulation by Chinese hamster ovary SecR cells similar to that of natural secretin (EC50 = 68 +/- 22 pM analogue and 95 +/- 25 pM secretin). It bound specifically and with high affinity (Ki = 5.0 +/- 1.1 nM) and covalently labeled the Mr = 57,000-62,000 secretin receptor. Cyanogen bromide cleavage of the receptor yielded a major labeled fragment of apparent Mr = 19,000 that shifted to Mr = 9,000 after deglycosylation. This was most consistent with either of two glycosylated domains within the amino-terminal tail of the receptor. Immunoprecipitation with antibody directed to epitope tags incorporated into each of the candidate domains established that the fragment at the amino terminus of the receptor was the site of labeling. This was further localized to the amino-terminal 30 residues of the receptor by additional proteolysis of this fragment with endoproteinase Lys-C. This provides the first direct demonstration of a contact between a secretin-like agonist and its receptor and will contribute a useful constraint to the modeling of this interaction.     

Temporal and spatial expression of the E5a protein during the differentiation-dependent life cycle of human papillomavirus type 31b

Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys, a chemotactic peptide that binds with high affinity to the chemoattractant receptor on granulocytes and monocytes, was labeled with 99mTc using the diaminedithiol (DADT) chelating system to coordinate the Tc. 99mTc labeling of the DADT-coupled peptide was accomplished in 84% overall yield (room temperature for 10 min) using [99mTc]glucoheptonate as the donor of prereduced Tc. HPLC analysis showed two major 99mTc-labeled peptide peaks, 99mTc-DADT-Pep-I and 99mTc-DADT-Pep-II, were obtained in a ratio of 1:0.85. Using an iodoacetamide-derivatized gel to remove unlabeled peptide from the 99mTc labeling mixtures, essentially no-carrier-added (nca) high-specific activity 99mTc-labeled chemotactic peptides were obtained. The 99Tc analogues of the peptides were synthesized (72% yield) in a similar fashion and correlated with 99mTc complexes I and II by HPLC. In vitro competitive receptor binding assays of the isolated 99Tc analogues were performed against the tritiated chemotactic peptide [3H]N-for-Met-Leu-Phe ([3H]fMLF) using isolated granulocytes. The 99Tc-derivatized peptides showed similar binding affinities to the chemoattractant receptor as the unlabeled Nalpha-for-Nle-Leu-Phe-Nle-Tyr-Lys. The nca 99mTc-labeled peptides gave high contrast images of experimental inflammation in rabbits without causing neutropenia. Thus, it is feasible to attach the Tc-DADT chelate to low-molecular weight receptor binding chemotactic peptides and retain substantial binding to the receptor. Chemotactic peptides labeled with 99mTc via the DADT ligand system have the potential for imaging focal sites of inflammation without toxic effects, an important consideration in the successful utilization of chemotactic peptide agonists.     

The amino-terminal region of the luteinizing hormone/choriogonadotropin receptor contacts both subunits of human choriogonadotropin. II. Photoaffinity labeling

The luteinizing hormone/choriogonadotropin receptor, a seven-transmembrane receptor, is composed of two equal halves, the N-terminal extracellular exodomain and the C-terminal membrane-associated endodomain. Unlike most seven-transmembrane receptors, the exodomain alone is responsible for high affinity hormone binding, whereas signal is generated in the endodomain. These physical separations of hormone-binding and receptor activation sites are attributed to unique mechanisms for hormone binding and receptor activation of this receptor and its subfamily members. However, the precise hormone contact sites in the exodomain are unclear. In the preceding article (Hong, S., Phang, T., Ji, I., and Ji, T. H. (1998) J. Biol. Chem. 273, 13835-13840), a region immediately downstream of the N terminus of the exodomain was shown to be crucial for hormone binding. To test if the region interacts with the hormone, human choriogonadotropin (hCG) was photoaffinity-labeled with a peptide mimic corresponding to Gly18-Tyr36 of the receptor. This peptide mimic specifically photoaffinity-labeled both the alpha- and beta-subunits of hCG. Interestingly, hCGalpha was preferentially labeled. On the other hand, denatured hCG was not labeled, and a mutant analog of the peptide failed to label hCG. Furthermore, the affinity labeling was UV-dependent and saturable, indicating the specificity of the photoaffinity labeling. Our results indicate that the region of the exodomain interacts with hCG and that the contact points are near both subunits of hCG. Particularly, the alternate residues (Leu20, Cys22, and Gly24) are crucial for hCG binding. In addition, the results underscore the fact that there is a crucial hormone contact site outside of the popularly believed primary hormone-binding site that is composed of Leu-rich repeats and is located in the middle of the exodomain. Our observations are crucial for understanding the molecular mechanism through which the initial high affinity hormone binding leads to receptor activation in the endodomain.     

The hepatic interleukin-6 receptor. Studies on its structure and regulation by phorbol 12-myristate 13-acetate-dexamethasone

Recombinant human 125I-interleukin-6 (IL-6) was cross-linked with the homobifunctional reagent disuccinimidyl suberate to human hepatoma cells (HepG2). Three recombinant human 125I-IL-6-containing complexes of apparent molecular masses of 100, 120, and 200 kDa were immunoprecipitated with specific antibodies to human IL-6 or to the 80-kDa IL-6 receptor subunit. We show by immunoprecipitation, peptide mapping, and by the use of a cleavable heterobifunctional cross-linker (Denny-Jaffe reagent) that different polypeptides are involved in the formation of the 100- and 120-kDa IL-6-containing complexes. The molecular compositions of the 100- and 120-kDa cross-linked complexes were identified. The 100-kDa complex consisted of one ligand and one IL-6 receptor subunit, glycoprotein 80 (gp80), whereas the 120-kDa complex was found to be composed of one ligand and a polypeptide which was immunoprecipitable with the monoclonal antibody AM64 directed against gp130. Exposure of HepG2 cells to phorbol 12-myristate 13-acetate (PMA) or PMA-dexamethasone led to an increase in the 80-kDa IL-6 receptor mRNA and functional receptor protein. Whereas treatment of HepG2 cells with PMA led to an increase in the formation of gp80.gp130.IL-6 complexes determined by cross-linking, no corresponding increase in high affinity binding sites was found. The existence of a third IL-6 receptor subunit present in limiting amounts on HepG2 cells is proposed to explain this discrepancy. Evidence is presented that the 80-kDa IL-6 receptor up-regulation by PMA-dexamethasone is caused by the depletion of protein kinase C since the protein kinase C inhibitor staurosporine mimics the effect of PMA-dexamethasone.     

Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site

Plasminogen activation by the urokinase-type plasminogen activator (uPA) is facilitated in the presence of cells expressing the glycolipid-anchored high-affinity receptor for uPA (denoted uPAR). Structures involved in the interaction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinity labeling [Ploug, M., Ostergaard, S., Hansen, L. B. L., Holm, A., and Dano, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe4 and Trp9 with either benzophenone or (trifluoromethyl)aryldiazirine rendered this peptide antagonist photoactivatable, and as a consequence, it incorporated covalently upon photolysis into either uPAR domain I or domain III depending on the actual position of the photophore in the sequence. The residues of uPAR specifically targeted by photoaffinity labeling were identified by matrix-assisted laser desorption mass spectrometry, NH2-terminal sequence analysis, and amino acid composition analysis after enzymatic fragmentation and HPLC purification. According to these data, the formation of the receptor-ligand complex positions Phe4 of the peptide antagonist very close to Arg53 and Leu66 in uPAR domain I and Trp9 of the antagonist in the vicinity of His251 in uPAR domain III. The gross molecular arrangement of the deduced receptor-ligand interface provides a rational structural basis for the observed requirement for the intact multidomain state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPAR domains I and III. In addition, these data suggest that the assembly of the composite ligand binding site in uPAR may resemble the homophilic interdomain dimerization of kappa-bungarotoxin, a structural homologue of the Ly-6/uPAR domain family.     

A role for Xenopus Frizzled 8 in dorsal development

The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction of either of these inhibitors of the substrates themselves with the catalytic domain of the enzymes has not been identified. IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photoaffinity labeling was progressively blocked by cGMP at concentrations higher than 10 microM, whereas cAMP or 5'-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5 catalytic site, including IBMX, cIMP, and beta-phenyl-1,N2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 microM, which compared favorably with an IC50 of 5 microM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in studies to map the catalytic site of PDE-5.     

Photoaffinity labeling of cytochrome P450 2B4: capture of active site heme ligands by a photocarbene

Spiro[adamantane-2,2'-diazirine], which produces adamantyl carbene upon photolysis, binds tightly to P450 2B4 (KS = 3.2 microM), giving a normal substrate binding difference spectrum. Irradiation of 2-[3H]adamantane diazirine at 365 nm in the presence of native, ferric P450 2B4 resulted in first-order photolysis (t1/2 = 1.8 min). The main product was 2-[3H]adamantanol, with about 6% of the radioactivity covalently bound to P450 2B4. With the ferrous carbonyl form of P450 2B4, 2-adamantanol production decreased and protein labeling increased to 12%. When ferric cyanide 2B4 was used, 2-adamantanecarbonitrile was formed in addition to 2-adamantanol. The nitrile appears to have resulted from capture of the iron-bound cyanide ligand by the carbene. The use of multiple cycles of photolysis increased the percentage of protein labeling to 76%. Photolabeling was inhibited by known 2B4 substrates and inhibitors. Also, N-demethylation of benzphetamine and generation of a substrate binding difference spectrum by benzphetamine were both inhibited stoichiometrically with the fraction of radiolabeled protein. The labeled protein was permanently converted to the high-spin state, as indicated by the characteristic change in the absorbance spectrum, demonstrating irreversible occupation of the substrate binding site by the adamantyl residue. Mild acid hydrolysis of radiolabeled 2B4 at the five Asp-Pro bonds generated a 2-kDa peptide which carried 78% of the radioactivity. These results are interpreted as the result of the active site carbene reacting by three competing pathways: capture of the heme sixth ligand to yield either 2-adamantanol or 2-adamantanecarbonitrile, capture of an unbound active site water molecule to yield adamantanol, and covalent attachment to a protein residue. Thus, the P450 2B4 active site appears to contain at least one unbound water molecule in addition to the heme aquo sixth ligand, even when substrate is present.