Stimulation of P-glycoprotein-mediated drug transport by prazosin and progesterone. Evidence for a third drug-binding site.

P-glycoprotein is a plasma membrane protein of mammalian cells that confers multidrug resistance by acting as a broad-specificity, ATP-dependent efflux transporter of diverse lipophilic neutral or cationic compounds. Previously, we identified two positively cooperative drug-binding sites of P-glycoprotein involved in transport [Shapiro, A. B. & Ling, V. (1997) Eur. J. Biochem. 250, 130-137]. The H site is selective for Hoechst 33342 and colchicine. The R site is selective for rhodamine 123 and anthracyclines. Substrate binding to one site stimulates transport by the other. In this paper, we show that prazosin and progesterone stimulate the transport of both Hoechst 33342 and rhodamine 123. Rhodamine 123 and prazosin (or progesterone) in combination stimulate Hoechst 33342 transport in an additive manner. In contrast, Hoechst 33342 and either prazosin or progesterone interfere with each other, so that the stimulatory effect of the combination on rhodamine 123 transport is less than that of each individually. Non-P-glycoprotein-specific effects of prazosin on membrane fluidity and permeability were excluded. These results indicate the existence of a third drug-binding site on P-glycoprotein with a positive allosteric effect on drug transport by the H and R sites. This allosteric site appears to be one of the sites of photoaffinity labeling of P-glycoprotein by [125I]iodoarylazidoprazosin [Safa, A. R., Agresti, M., Bryk, D. & Tamai, I. (1994) Biochemistry 33, 256-265] and is likely not to be capable of drug transport.     

Cytoplasmic orientation and two-domain structure of the multidrug transporter, P-glycoprotein, demonstrated with sequence-specific antibodies

The predicted cytoplasmic orientation and two-domain structure of the multidrug efflux pump P-glycoprotein were demonstrated with sequence-specific antibodies. We synthesized peptides corresponding to amino acid residues, Glu393-Lys408 (anti-P) and Leu1206-Thr1226 (anti-C) in P-glycoprotein from human mdr1 cDNA and used these peptides to produce polyclonal antibodies. From the primary structure of P-glycoprotein, and anti-C antibody is expected to recognize another position, Leu561-Thr581, in the duplicate structure of P-glycoprotein, but anti-P recognizes only one site. These antibodies bind to multidrug-resistant cells (KB-C2) with permeabilized plasma membrane but do not bind to nonpermeabilized KB-C2 cells or parental KB cells, supporting the predicted cytoplasmic orientation of these sequences. With immunoblotting of the membrane fractions from KB-C2 cells, a major 140-kDa polypeptide of the P-glycoprotein was detected with both anti-P and anti-C. Two minor polypeptides with molecular mass of 95 and 55 kDa were also detected. When membrane vesicles were digested mildly with trypsin, the amount of these two polypeptides increased. Anti-P detected only the 95-kDa polypeptide, and anti-C detected both 95- and 55-kDa polypeptides. Achromobacter lyticus protease I (lysyl endopeptidase) and Staphylococcus aureus V8 protease also produced two polypeptides with similar molecular weights. Absorption into lectin-agarose beads and labeling with [3H]glucosamine indicated that the 95-kDa polypeptide was glycosylated but that the 55-kDa polypeptide was not. These two polypeptides as well as P-glycoprotein were photoaffinity-labeled with a calcium channel blocker, [3H]azidopine, but most of the label was found in the 55-kDa polypeptide. The yield of labeled fragments from membrane vesicles photolabeled after digestion with trypsin was similar to that from membrane vesicles digested with trypsin after photolabeling. These data indicate 1) that the 95-kDa polypeptide is the fragment corresponding to the amino-terminal half of P-glycoprotein containing sugar chains; 2) that the 55-kDa polypeptide is the carboxyl-terminal half which was mainly labeled with [3H]azidopine; and 3) that P-glycoprotein has a relatively rigid structure with a small number of protease-sensitive sites and its global structure is not destroyed by tryptic cleavage.     

Intrinsic fluorescence of the P-glycoprotein multidrug transporter: sensitivity of tryptophan residues to binding of drugs and nucleotides

P-glycoprotein is a member of the ATP binding cassette family of membrane proteins, and acts as an ATP-driven efflux pump for a diverse group of hydrophobic drugs, natural products, and peptides. The side chains of aromatic amino acids have been proposed to play an important role in recognition and binding of substrates by P-glycoprotein. Steady-state and lifetime fluorescence techniques were used to probe the environment of the 11 tryptophan residues within purified functional P-glycoprotein, and their response to binding of nucleotides and substrates. The emission spectrum of P-glycoprotein indicated that these residues are present in a relatively nonpolar environment, and time-resolved experiments showed the existence of at least two lifetimes. Quenching studies with acrylamide and iodide indicated that those tryptophan residues predominantly contributing to fluorescence emission are buried within the protein structure. Only small differences in Stern-Volmer quenching constants were noted on binding of nucleotides and drugs, arguing against large changes in tryptophan accessibility following substrate binding. P-glycoprotein fluorescence was highly quenched on binding of fluorescent nucleotides, and moderately quenched by ATP, ADP, and AMP-PNP, suggesting that the site for nucleotide binding is located relatively close to tryptophan residues. Drugs, modulators, hydrophobic peptides, and nucleotides quenched the fluorescence of P-glycoprotein in a saturable fashion, allowing estimation of dissociation constants. Many compounds exhibited biphasic quenching, suggesting the existence of multiple drug binding sites. The quenching observed for many substrates was attributable largely to resonance energy transfer, indicating that these compounds may be located close to tryptophan residues within, or adjacent to, the membrane-bound domains. Thus, the regions of P-glycoprotein involved in nucleotide and drug binding appear to be packed together compactly, which would facilitate coupling of ATP hydrolysis to drug transport.     

Equilibrium, kinetic and photoaffinity labeling studies of daunomycin binding to P-glycoprotein-containing membranes of multidrug-resistant Chinese hamster ovary cells

The binding of daunomycin and its Bolton-Hunter derivative iodomycin to plasma membranes isolated from multidrug-resistant Chinese hamster ovary cells (CHO B30) and their drug-sensitive parents (B1) was investigated. The thermodynamics and kinetics of equilibrium binding monitored by fluorescence titrations and temperature-jump relaxation spectrometry were compared with the specificity of covalent photolabeling with [3H]daunomycin and [125I]iodomycin. The facts that the uptake of anthracycline from aqueous solution into the CHO membranes was not accompanied by any substantial increase of fluorescence anisotropy nor by any spectral shift of the fluorescence emission spectrum and that the partition ratio into the membrane was 20-30-fold higher when compared to a lecithin bilayer, provided evidence that the non-covalent drug binding sites are constituted by polar protein domains without any substantial contribution from the surrounding lipids. Photoaffinity labeling with nanomolar concentrations of anthracycline and equilibrium binding curves independently showed that a 150-170-kDa plasma membrane glycoprotein (P-glycoprotein), whose overexpression is the major difference between B1 and B30 membranes, provides the binding sites of highest affinity for daunomycin and iodomycin (K approximately equal to 4 x 10(7) M-1). Comparison of photolabeling and equilibrium data suggested that the same binding sites on P-glycoprotein were most probably being monitored. The photolabeling of P-glycoprotein by iodomycin was inhibited in a dose-dependent manner by other compounds to which multi-drug-resistant cells are either resistant or collaterally sensitive with the following orders of effectiveness: vinblastine greater than verapamil greater than nitrendipine greater than daunomycin much greater than colchicine. Temperature-jump experiments covering the time range of 1 microseconds to 1 s revealed a single concentration-dependent relaxation time of 10-30 microseconds. The association of daunomycin with its binding sites in the membranes was found to be a diffusion-controlled process with kon rates of 2-4 X 10(9) M-1 s-1. Therefore, the selectivity of drug binding was entirely reflected in the dissociation rates.     

P-Glycoprotein kinetics measured in plasma membrane vesicles and living cells

P-glycoprotein (MDR1, ABCB1) is an ATP-dependent efflux transporter of a large variety of compounds. To understand P-glycoprotein in more detail, it is important to elucidate its activity in the cellular ensemble as well as in plasma membrane vesicles (under conditions where other ATP dependent proteins are blocked). We measured P-glycoprotein activity in inside-out vesicles formed from plasma membranes of MDR1-transfected mouse embryo fibroblasts (NIH-MDR1-G185) for comparison with previous measurements of P-glycoprotein activity in living NIH-MDR1-G185 cells. In plasma membrane vesicles activity was measured by monitoring phosphate release upon ATP hydrolysis and in living cells by monitoring the extracellular acidification rate upon ATP synthesis via glycolysis. P-glycoprotein was stimulated as a function of the concentration with 19 structurally different drugs, including local anesthetics, cyclic peptides, and cytotoxic drugs. The concentrations of half-maximum P-glycoprotein activation, K1, were identical in inside-out plasma membrane vesicles and in living cells and covered a broad range of concentrations (K1 approximately (10(-8)-10(-3)) M). The influence of the pH, drug association, and vesicle aggregation on the concentration of half-maximum P-glycoprotein activation was investigated. The turnover numbers in plasma membrane vesicles and in living cells were also approximately identical if the latter were measured in the presence of pyruvate. However, in the absence of pyruvate they were higher in living cells. The rate of ATP hydrolysis/ATP synthesis decreased exponentially with decreasing free energy of drug binding from water to the transporter, DeltaG0(tw)(1) (or increasing binding affinity). This suggests that drug release from the transmembrane domains has to occur before ATP is hydrolyzed for resetting the transporter.     

The role of hydrogen bond acceptor groups in the interaction of substrates with Pdr5p, a major yeast drug transporter

The yeast ABC (ATP-binding cassette protein) multidrug transporter Pdr5p transports a broad spectrum of xenobiotic compounds, including antifungal and antitumor agents. Previously, we demonstrated that substrate size is an important factor in substrate-transporter interaction and that Pdr5p has at least three substrate-binding sites. In this study, we use a combination of whole cell transport assays and photoaffinity labeling of Pdr5p with [(125)I]iodoarylazidoprazosin in purified plasma membrane vesicles to study the behavior of two series of novel substrates: trityl (triphenylmethyl) and carbazole derivatives. The results indicate that site 2, defined initially by tritylimidazole efflux, requires at least a single hydrogen bond acceptor group (electron pair donor). In contrast, complete inhibition of rhodamine 6G efflux and [(125)I]iodoarylazidoprazosin binding at site 1 requires substrates with three electronegative groups. Carbazole and trityl substrates with two groups show saturating, incomplete inhibition at this site. This type of inhibition is frequently observed in bacterial multidrug-binding proteins that use a pocket with multiple binding sites. The presence of multiple sites with different requirements for substrate-Pdr5p interaction may explain the broad specificity of xenobiotic compounds transported by this protein.     

Unique substrate recognition by botulinum neurotoxins serotypes A and E

Botulinum neurotoxins (BoNTs) are zinc proteases that cleave SNARE proteins to elicit flaccid paralysis by inhibiting the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. There are seven serotypes of BoNT, termed A-G. BoNT serotype A and serotype E cleave SNAP25 at residues 197-198 and 180-181, respectively. Unlike other zinc proteases, the BoNTs recognize extended regions of SNAP25 for cleavage. The basis for this extended substrate recognition and specificity is unclear. Saturation mutagenesis and deletion mapping identified residues 156-202 of SNAP25 as the optimal cleavage domain for BoNT/A, whereas the optimal cleavage domain for BoNT/E was shorter, comprising residues 167-186 of SNAP25. Two sub-sites were resolved within each optimal cleavage domain, which included a recognition or active site (AS) domain that contained the site of cleavage and a binding (B) domain, which contributed to substrate affinity. Within the AS domains, the P1', P3, and P5 sites of SNAP25 contributed to scissile bond cleavage by LC/A, whereas the P1' and P2 sites of SNAP25 contributed to scissile bond cleavage by LC/E. These studies provide insight into the development of strategies for small molecule inhibitors of the BoNTs.     

Caveolin scaffolding region and the membrane binding region of SRC form lateral membrane domains

Formation of domains by the membrane binding motifs of caveolin and src were studied in large unilamellar vesicles using fluorescence digital imaging microscopy. Caveolin, a major structural protein of caveolae, contains a scaffolding region (residues 82-101) that contributes to the binding of the protein to the plasma membrane. A caveolin peptide (82-101) corresponding to this scaffolding region induced the formation of membrane domains enriched in the acidic lipids phosphatidylserine and phosphatidylinositol-4,5-bisphosphate. Cholesterol, another predominant component of caveolae, was also enriched in these domains. Caveolae also contain many different signaling molecules including src family tyrosine kinases. Src proteins bind to the plasma membrane via a N-terminal myristate chain and a cluster of basic residues that can interact electrostatically with negatively charged lipids. A peptide corresponding to the src membrane binding motifs (residues myr-2-19) sequestered acidic lipids into lateral membrane domains. Both the src and the caveolin peptides colocalized together with acidic lipids in the domains. Control experiments show the domains are not the result of vesicle aggregation. Two-photon fluorescence correlation spectroscopy experiments suggest diffusion in the domains was slower, but the domains were dynamic. Protein kinase C phosphorylated src in its N-terminal membrane binding region; however, the caveolin scaffolding peptide inhibited this activity. Consequently, protein-induced membrane domains may affect cell signaling by organizing signal transduction components within the membrane and changing reaction rates.     

Interaction of LDS-751 with P-glycoprotein and mapping of the location of the R drug binding site

One cause of multidrug resistance is the overexpression of P-glycoprotein, a 170 kDa plasma membrane ABC transporter, which functions as an ATP-driven efflux pump with broad specificity for hydrophobic drugs, peptides, and natural products. The protein appears to interact with its substrates within the membrane environment. Previous reports suggested the existence of at least two binding sites, possibly overlapping and displaying positively cooperative interactions, termed the H and R sites for their preference for Hoechst 33342 and rhodamine 123, respectively. In this work, we have used several fluorescence approaches to characterize the molecular interaction of purified P-glycoprotein (Pgp) with the dye LDS-751, which is proposed to bind to the R site. A 50-fold enhancement of LDS-751 fluorescence indicated that the protein binding site was located in a hydrophobic environment, with a polarity lower than that of chloroform. LDS-751 bound with sub-micromolar affinity (K(d) = 0.75 microM) and quenched P-glycoprotein intrinsic Trp fluorescence by 40%, suggesting that Trp emitters are probably located close to the drub-binding regions of the transporter and may interact directly with the dye. Using a FRET approach, we mapped the possible locations of the LDS-751 binding site relative to the NB domain active sites. The R site appeared to be positioned close to the membrane boundary of the cytoplasmic leaflet. The location of both H and R drug binding sites is in agreement with the idea that Pgp may operate as a drug flippase, moving substrates from the inner leaflet to the outer leaflet of the plasma membrane.     

Location of the rhodamine-binding site in the human multidrug resistance P-glycoprotein

The human multidrug resistance P-glycoprotein (P-gp) pumps a wide variety of structurally diverse compounds out of the cell. It is an ATP-binding cassette transporter with two nucleotide-binding domains and two transmembrane (TM) domains. One class of compounds transported by P-gp is the rhodamine dyes. A P-gp deletion mutant (residues 1-379 plus 681-1025) with only the TM domains retained the ability to bind rhodamine. Therefore, to identify the residues involved in rhodamine binding, 252 mutants containing a cysteine in the predicted TM segments were generated and reacted with a thiol-reactive analog of rhodamine, methanethiosulfonate (MTS)-rhodamine. The activities of 28 mutants (in TMs 2-12) were inhibited by at least 50% after reaction with MTS-rhodamine. The activities of five mutants, I340C(TM6), A841C(TM9), L975C(TM12), V981C(TM12), and V982C(TM12), however, were significantly protected from inhibition by MTS-rhodamine by pretreatment with rhodamine B, indicating that residues in TMs 6, 9, and 12 contribute to the binding of rhodamine dyes. These results, together with those from previous labeling studies with other thiol-reactive compounds, dibromobimane, MTS-verapamil, and MTS-cross-linker substrates, indicate that common residues are involved in the binding of structurally different drug substrates and that P-gp has a common drug-binding site. The results support the "substrate-induced fit" hypothesis for drug binding.     

Molecular determinants of ligand binding to the human melanocortin-4 receptor

To elucidate the molecular basis for the interaction of ligands with the human melanocortin-4 receptor (hMC4R), agonist structure-activity studies and receptor point mutagenesis were performed. Structure-activity studies of [Nle(4), D-Phe(7)]-alpha-melanocyte stimulating hormone (NDP-MSH) identified D-Phe7-Arg8-Trp9 as the minimal NDP-MSH fragment that possesses full agonist efficacy at the hMC4R. In an effort to identify receptor residues that might interact with amino acids in this tripeptide sequence 24 hMC4R transmembrane (TM) residues were mutated (the rationale for choosing specific receptor residues for mutation is outlined in the Results section). Mutation of TM3 residues D122 and D126 and TM6 residues F261 and H264 decreased the binding affinity of NDP-MSH 5-fold or greater, thereby identifying these receptor residues as sites potentially involved in the sought after ligand-receptor interactions. By examination of the binding affinities and potencies of substituted NDP-MSH peptides at receptor mutants, evidence was found that core melanocortin peptide residue Arg8 interacts at a molecular level with hMC4R TM3 residue D122. TM3 mutations were also observed to decrease the binding of hMC4R antagonists. Notably, mutation of TM3 residue D126 to alanine decreased the binding affinity of AGRP (87-132), a C-terminal derivative of the endogenous melanocortin antagonist, 8-fold, and simultaneous mutations D122A/D126A completely abolished AGRP (87-132) binding. In addition, mutation of TM3 residue D122 or D126 decreased the binding affinity of hMC4R antagonist SHU 9119. These results provide further insight into the molecular determinants of hMC4R ligand binding.     

Structure of the membrane reconstituted transmembrane-juxtamembrane peptide EGFR(622-660) and its interaction with Ca2+/calmodulin

The transmembrane (TM) and juxtamembrane (JM) regions of the epidermal growth factor receptor (EGFR) couple ligand binding in the extracellular domain to activation of the kinase domain. Solid-state NMR and polarized FTIR measurements of peptides corresponding to the TM plus JM regions of EGFR (residues 622-660) reconstituted in model phospholipid membranes are presented to address the role of the short cytoplasmic JM sequence (residues 645-660) in regulating EGFR activity. We show that the TM domain is helical with a transition to non-helical structure at the TM-JM boundary. Fluorescence measurements indicate that the JM region of EGFR(622-660) binds to the membrane surface and that binding can be reversed by the addition of the complex of Ca2+ and calmodulin. Together these data support models suggesting the cytoplasmic JM region of EGFR plays an active role in regulating receptor activity.     

Proximity of bound Hoechst 33342 to the ATPase catalytic sites places the drug binding site of P-glycoprotein within the cytoplasmic membrane leaflet

The P-glycoprotein multidrug transporter carries out ATP-driven cellular efflux of a wide variety of hydrophobic drugs, natural products, and peptides. Multiple binding sites for substrates appear to exist, most likely within the hydrophobic membrane spanning regions of the protein. Since ATP hydrolysis is coupled to drug transport, the spatial relationship of the drug binding sites relative to the ATPase catalytic sites is of considerable interest. We have used a fluorescence resonance energy transfer (FRET) approach to estimate the distance between a bound substrate and the catalytic sites in purified P-glycoprotein. The fluorescent dye Hoechst 33342 (H33342), a high-affinity P-glycoprotein substrate, bound to the transporter and acted as a FRET donor. H33342 showed greatly enhanced fluorescence emission when bound to P-glycoprotein, together with a substantial blue shift, indicating that the drug binding site is located in a nonpolar environment. Cys428 and Cys1071 within the catalytic sites of P-glycoprotein were covalently labeled with the acceptor fluorophore NBD-Cl (7-chloro-4-nitrobenz-2-oxa-1,3-diazole). H33342 fluorescence was highly quenched when bound to NBD-labeled P-glycoprotein relative to unlabeled protein, indicating that FRET takes place from the bound dye to NBD. The distance separating the bound dye from the NBD acceptor was estimated to be approximately 38 A. Transition-state P-glycoprotein with the complex ADP*orthovanadate*Co2+ stably trapped at one catalytic site bound H33342 with similar affinity, and FRET measurements led to a similar separation distance estimate of 34 A. Since previous FRET studies indicated that a fluorophore bound within the catalytic site was positioned 31-35 A from the interfacial region of the bilayer, the H33342 binding site is likely located 10-14 A below the membrane surface, within the cytoplasmic leaflet of the membrane, in both resting-state and transition-state P-glycoprotein.     

Degradation of a signal peptide by protease IV and oligopeptidase A.

The degradation of the prolipoprotein signal peptide in vitro by membranes, cytoplasmic fraction, and two purified major signal peptide peptidases from Escherichia coli was followed by reverse-phase liquid chromatography (RPLC). The cytoplasmic fraction hydrolyzed the signal peptide completely into amino acids. In contrast, many peptide fragments accumulated as final products during the cleavage by a membrane fraction. Most of the peptides were similar to the peptides formed during the cleavage of the signal peptide by the purified membrane-bound signal peptide peptidase, protease IV. Peptide fragments generated during the cleavage of the signal peptide by protease IV and a cytoplasmic enzyme, oligopeptidase A, were identified from their amino acid compositions, their retention times during RPLC, and knowledge of the amino acid sequence of the signal peptide. Both enzymes were endopeptidases, as neither dipeptides nor free amino acids were formed during the cleavage reactions. Protease IV cleaved the signal peptide predominantly in the hydrophobic segment (residues 7 to 14). Protease IV required substrates with hydrophobic amino acids at the primary and the adjacent substrate-binding sites, with a minimum of three amino acids on either side of the scissile bond. Oligopeptidase A cleaved peptides (minimally five residues) that had either alanine or glycine at the P'1 (primary binding site) or at the P1 (preceding P'1) site of the substrate. These results support the hypothesis that protease IV is the major signal peptide peptidase in membranes that initiates the degradation of the signal peptide by making endoproteolytic cuts; oligopeptidase A and other cytoplasmic enzymes further degrade the partially degraded portions of the signal peptide that may be diffused or transported back into the cytoplasm from the membranes.     

Partial cleavage mapping of the cytoskeletal protein vinculin. Antibody and talin binding sites.

Vinculin is a 1066-amino acid protein found at several types of actin-membrane junction. To locate sites of interest in the primary structure, a map was derived using partial cleavage reactions. Of several different types of cleavage tested, the most useful was the 5-5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) reaction which cuts at cysteine residues. About 30 well defined fragments were obtained from vinculin, and several methods were used to locate these products in the sequence. Comparison of the peptides generated from whole vinculin with those from the 90-kDa amino-terminal proteolytic fragment revealed which originated there. The use of [14C]cyanide in conjunction with DTNB showed which peptides contained the original amino terminus. Secondary cleavage with N-chlorosuccinimide, a tryptophan-specific reagent, helped locate fragments, although it led to apparent increases in molecular weight of the products. These experiments revealed the location of 10 of the major DTNB fragments on the sequence. This map was used to locate binding sites. The site of interaction between vinculin and the focal contact protein talin was mapped by binding labeled talin to the separated fragments. The binding site was found to be in the amino-terminal 325 amino acids. The binding site of a commercially obtained monoclonal antivinculin antibody was mapped using Western blotting of cleaved vinculin. It proved to bind in the central area of the molecule between amino acid residues 545 and 737. Thus the cysteine cleavage reaction products provide a map of general utility for locating features on the vinculin molecule.     

Molecular identification of the human melanocortin-2 receptor responsible for ligand binding and signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.     

The application of an improved solid phase synthetic technique to the delineation of an antigenic site of apolipoprotein A-II

Previous studies have shown that the antigenic sites of human plasma high-density apolipoprotein A-II (apoA-II) are separate from their lipid-binding determinants in human high density lipoproteins (HDL). A specific radioimmunoassay has shown that three distinct antigenic sites are located in residues 4-23, 31-46, and 56-77; these studies suggested that an antigenic site might be restricted to residues 60-77 in the 56-77 fragment. To further delineate this site, we have developed a solid phase radioimmunoassay technique using an improved solid support on which selected sequences of peptides were synthesized, deprotected with HF, and the resulting peptidyl-resins tested for their capability of binding purified 125I-anti-apoA-II antibodies. Amino acid analyses and solid phase sequence analyses were performed to verify the sequence of the synthetic peptide on the solid support. Using this technique, 125I-anti-apoA-II antibodies had achieved 50% of maximal binding when residues 61-77 were attached to the solid support. The maximal binding was achieved by the addition of one more residue, Leu60, thus confirming our suggestion that a major antigenic site is located in residues 60-77. The binding to the peptidyl-resin was inhibited by a synthetic fragment corresponding to residues 60-77 indicating that the antibodies were specifically bound to the resin.     

Structure and ligand binding of the soluble domain of a Thermotoga maritima membrane protein of unknown function TM1634

As a part of the Joint Center for Structural Genomics (JCSG) biological targets, the structures of soluble domains of membrane proteins from Thermotoga maritima were pursued. Here, we report the crystal structure of the soluble domain of TM1634, a putative membrane protein of 128 residues (15.1 kDa) and unknown function. The soluble domain of TM1634 is an alpha-helical dimer that contains a single tetratrico peptide repeat (TPR) motif in each monomer where each motif is similar to that found in Tom20. The overall fold, however, is unique and a DALI search does not identify similar folds beyond the 38-residue TPR motif. Two different putative ligand binding sites, in which PEG200 and Co(2+) were located, were identified using crystallography and NMR, respectively.     

The extracellular loop between TM5 and TM6 of P-glycoprotein is required for reactivity with monoclonal antibody UIC2.

P-glycoprotein (P-gp), encoded by the MDR1 gene, is a plasma membrane transporter which confers resistance to many chemotherapeutic drugs. Monoclonal antibodies raised against P-gp have been used as tools to study P-gp topology and activity. Monoclonal antibody UIC2 recognizes a functional conformation of P-gp on the cell surface and blocks P-gp-mediated drug transport. Knowledge about the UIC2 epitope and the mechanism of its inhibitory effects may be helpful for understanding P-gp structure and developing P-gp inhibitors. In the present work, using several chimeras of MDR1 and MDR2, we found that the native sequence of the predicted extracellular loop between transmembrane domains (TM) 5 and 6 of P-gp is necessary, but not sufficient, for UIC2 reactivity. In addition, UIC2 reactivity is also affected by mutations in TM6, a region known to be involved in interactions of P-gp with substrates. These observations suggest that residues in the extracellular loop between TM5 and TM6 are directly involved in the display of the UIC2 epitope. Since TM6 has been shown to be actively involved in drug transport process, the proximity of this region to TM6 may help to explain why UIC2 binding is sensitive to the functional state of P-gp and why binding of UIC2 inhibits P-gp-mediated drug transport.     

Characterization of endothelin-converting enzyme-2. Implication for a role in the nonclassical processing of regulatory peptides

Most neuroendocrine peptides are generated by proteolysis of the precursors at basic residue cleavage sites. Prohormone convertases belonging to the subtilisin family of serine proteases are primarily responsible for processing at these "classical sites." In addition to the classical cleavages, a subset of bioactive peptides is generated by processing at "nonclassical" sites. The proteases responsible for these cleavages have not been well explored. Members of several metalloprotease families have been proposed to be involved in nonclassical processing. Among them, endothelin-converting enzyme-2 (ECE-2) is a good candidate because it exhibits a neuroendocrine distribution and an acidic pH optimum. To examine the involvement of this protease in neuropeptide processing, we purified the recombinant enzyme and characterized its catalytic activity. Purified ECE-2 efficiently processes big endothelin-1 to endothelin-1 by cleavage between Trp(21) and Val(22) at acidic pH. To characterize the substrate specificity of ECE-2, we used mass spectrometry with a panel of 42 peptides as substrates to identify the products. Only 10 of these 42 peptides were processed by ECE-2. A comparison of residues around the cleavage site revealed that ECE-2 exhibits a unique cleavage site selectivity that is related to but distinct from that of ECE-1. ECE-2 tolerates a wide range of amino acids in the P1-position and prefers aliphatic/aromatic residues in the P1'-position. However, only a small fraction of the aliphatic/aromatic amino acid-containing sites were cleaved, indicating that there are additional constraints beyond the P1- and P1'-positions. The enzyme is able to generate a number of biologically active peptides from peptide intermediates, suggesting an important role for this enzyme in the biosynthesis of regulatory peptides. Also, ECE-2 processes proenkephalin-derived bovine adrenal medulla peptides, and this processing leads to peptide products known to have differential receptor selectivity. Finally, ECE-2 processes PEN-LEN, an endogenous inhibitor of prohormone convertase 1, into products that do not inhibit the enzyme. Taken together, these results are consistent with an important role for ECE-2 in the processing of regulatory peptides at nonclassical sites.