Richard Leduc | Université de Sherbrooke (University of Sherbrooke) (original) (raw)

Papers by Richard Leduc

Journal of Biological Chemistry, 2002

Activation of G protein-coupled receptors by agonists involves significant movement of transmembr... more Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TM) following binding of agonist. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which constitutes a water-accessible crevice surrounded by the seven TM helices. Using the substituted cysteine accessibility method, we initially identified those residues within the seventh transmembrane domain (TM7) of wild type angiotensin II type 1 (AT 1 ) receptor that contribute to forming the binding site pocket. We have substituted successively TM7 residues ranging from Ile 276 to Tyr 302 to cysteine. Treatment of A277C, V280C, T282C, A283C, I286C, A291C, and F301C mutant receptors with the charged sulfhydryl-specific alkylating agent MTSEA significantly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT 1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was greatly reduced for TM7 reporter cysteines engineered in a constitutively active mutant of the AT 1 receptor. Our data suggest that upon activation, TM7 of the AT 1 receptor goes through a pattern of helical movements that results in its distancing from the binding pocket per se. These studies support accumulating evidence whereby elements of TM7 of class A GPCRs promote activation of the receptor through structural rearrangements.

Canadian Journal of Physiology and Pharmacology, 2002

We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards t... more We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.

Biochemistry, 2002

The human angiotensin II type 1 receptor (hAT 1 ) was photolabeled with a high-affinity radiolabe... more The human angiotensin II type 1 receptor (hAT 1 ) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, 125 I-[Sar 1 , Val 5 , p-Benzoyl-L-phenylalanine 8 ]AngII ( 125 I-[Sar 1 ,Bpa 8 ]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT 1 . Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe 293 and Asn 294 within the seventh transmembrane domain of the hAT 1 . Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N-and C-terminal proximity of those residues 293 and 294 of the hAT 1 produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT 1 receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (

Angiotensin Protocols, 2000

Photoaffinity labeling is a useful method to covalently bind two interacting moieties whether the... more Photoaffinity labeling is a useful method to covalently bind two interacting moieties whether they be substrate and enzyme or ligand and receptor. Irreversibly labeling any particular molecule is a practical way of detecting the latter throughout the course of a characterization or a purification procedure.

Journal of Medicinal Chemistry, 2014

We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homol... more We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homologous proteases matriptase and matriptase-2 across subpockets using docking simulations. We observed that the farther away a subpocket is located from the catalytic site, the more pronounced its role in selectivity. As a result of our exhaustive virtual screening, we biochemically validated novel potent and selective inhibitors of both enzymes.

Pharmacogenetics and Genomics, 2010

Molecular Pharmacology, 2008

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that a... more Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97(2.50) as well as residues Glu147(3.49), Arg148(3.50), and Tyr149(3.51) of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97(2.50)A, R148(3.50)A, and R148(3.50)H abolished the ability of UT to activate phospholipase C, whereas mutations E147(3.49)A and Y149(3.51)A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148(3.50)A and R148(3.50)H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Galpha(q/11)-independent transactivation of EGFR. The D97(2.50)A, R148(3.50)A, and R148(3.50)H mutants did not readily internalize and did not promote translocation or colocalize with beta-arrestin2-GFP. Finally, the agonist-induced internalization of the E147(3.49)A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp(2.50) residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Galpha(q/11)-dependent and -independent events can occur.

Molecular Pharmacology, 2007

The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predi... more The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predicted to undergo conformational changes after agonist binding, was investigated using the substituted-cysteine accessibility method. Each residue in the Lys240 -Leu265 fragment was mutated, one at a time, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydrylspecific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). This treatment led to a significant reduction in binding of 125 I-[Sar 1 ,Ile 8 ]AngII to the F249C, H256C, T260C, and V264C mutant receptors, suggesting that these residues orient themselves within the water-accessible binding pocket of the AT 1 receptor. It is noteworthy that this pattern of acquired MTSEA sensitivity was altered for TMD6 cysteines engineered in a constitutively active AT 1 receptor. Indeed, mutant F249C was insensitive to MTSEA treatment, whereas the sensitivity of mutant V264C decreased. Under these conditions, one other mutant, F261C, was found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT 1 receptor causes TMD6 to pivot. This movement moves the top (extracellular side) of TMD6 toward the binding pocket and simultaneously distances the bottom (intracellular side) away from the binding pocket. Using this approach, we identified key elements within TMD6 that contribute to the activation of class A GPCRs through structural rearrangements.

The Journal of biological chemistry, 2015

Biased signalling represents the ability of G protein-coupled receptors (GPCRs) to engage distinc... more Biased signalling represents the ability of G protein-coupled receptors (GPCRs) to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 receptor (AT1), a prototypical class A GPCR, can activate various effectors upon stimulation with the endogenous ligand AngII including the Gq/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated to distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations (MD) were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway) and the D74N-AT1 receptor (biased for the beta-arrestin 1 and 2 pathways) in their apo forms and in complex with AngII. The MD simulations of the AngII-WT-AT1, N111G-AT1 and AngII-N111G-AT1 receptors revealed specific structural rearrangem...

Nature Communications, 2015

Molecular pharmacology, Jan 25, 2015

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the act... more The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein-coupled receptor. The AT1 receptor engages and activates several signalling pathways, including heterotrimeric G proteins Gq and G12, as well as the ERK1/2 pathway. Additionally, following stimulation, β-arrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favour the activation of some signalling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of 6 different signalling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4 and 8, we sough...

Journal of Peptide Science, 2007

Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protei... more Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT 1 has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT 1 receptor and two representative Met mutants (H256M-hAT 1 and F293M-hAT 1 ) from the iterative mutagenesis study were photolabelled with the benzophenoneligand 125 I-[Sar 1 , Bpa 8 ]AngII at temperatures ranging from −15°C to 37°C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT 1 and F293M-hAT 1 across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT 1 was observed. At −15°C, H256M labelling was identical to that of hAT 1 , indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37°C labelling occurred almost exclusively at mutated residue H256M-hAT 1 in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures.

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 p... more Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene produc0 contained an elevated level of a membrane-associated endoproteolytic activ- ity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (fu- fin) shared many properties with Kex2p including ac- tivity at pH

The International Journal of Biochemistry & Cell Biology, 2009

a b s t r a c t ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in... more a b s t r a c t ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR 46 , RRR 69 and RRRRR 261 ) suggested that proADAMTS5 processing occurs after Arg 261 . That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

PloS one, 2014

Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine prot... more Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P') sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 indi...

European Journal of Biochemistry, 1997

Production of endothelin-1 is thought to be a three-step process consisting of an initial proteol... more Production of endothelin-1 is thought to be a three-step process consisting of an initial proteolytic cleavage of the proendothelin-1 precursor to big endothelin-I-Lys-Arg, C-terminal trimming by a carboxypeptidase and further processing of the big endothelin-I peptide to endothelin-1 by endothelinconverting enzyme (ECE). To further clarify the mechanism of processing in the biosynthesis of endothelin-1, we introduced a point mutation into endothelin-I cDNA to replace the Arg in the -4 position of the recognition motifs of furin-like convertase in human preproendothelin-1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in Chinese hamster ovary (CH0)-K1 cells, they failed to be processed at the mutated processing signal, suggesting that the Arg-Ser-Lys-Arg motifs of preproendothelin-1 are recognized by CHO-K1 furin-like convertase. Co-transfection with ECE-1 cDNA revealed that cleavage at ArgS2 is not essential for cleavage by ECE-1, but that cleavage at Arg92 is critical. Although a high-molecular-mass form of endothelin-1 is produced by processing by ECE-1 without cleavage at Arg52, it did not evoke Ca2+ transient in endothelin,-receptor-expressing cells. In conclusion, prior cleavage at Arg92 by furin-like convertase is absolutely necessary for cleavage by ECE-1 at Trp73 to produce mature endothelin-1 .

Prostaglandins, Leukotrienes and Essential Fatty Acids, 1998

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory ... more Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases. NSAIDs inhibit cyclooxygenase (COX), the rate limiting enzyme responsible for the conversion of arachidonic acid into prostaglandins. Recent studies have shown the existence of two isoforms of cyclooxygenase: COX-l, now often referred to as the constitutive form, and COX-2, an inducible form which is the major isoenzyme involved in prostaglandin synthesis in inflammation and other pathological situations. Since inhibition of prostaglandin production in tissues where they play a physiological role leads to important side effects, a COX-2 preferential inhibitor would present therapeutical advantages. In the present study, we evaluated the inhibitory properties of cyclooxygenase inhibitors on human COX-1 and COX-2 using a heterologous expression system. We investigated instantaneous inhibition and pre-incubation inhibition as well as time recovery of cyclooxygenase activity assays with the aid of four NSAIDs: mefenamic acid, indomethacin, aspirin and NS-398. Our results demonstrate that instantaneous inhibition assays have little correlation with clinical results. Inhibition assays using pre-incubation with the drugs tested, however, more closely resemble the data from in vivo studies. Cyclooxygenase recovery assays enabled better characterization of simple competitive inhibitors, competitive reversible time-dependent inhibitors and irreversible time-dependent inhibitors. The data illustrate the usefulness of our system in allowing a better determination of the pharmacological characteristics of NSAIDs as well as permitting a comparison among different drugs.

Proceedings of the National Academy of Sciences, 1991

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrineprecursor... more Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrineprecursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursorprocessing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processingdeficient BSC-40 cells cleaved native mouse POMC at the -Lys-Argsites flanking corticotropin. The -Lys-Argwithin 13-lipotropin was less efficiently cleaved to release f-endorphin.

Pharmacogenetics and Genomics, 2010

Journal of Biological Chemistry, 2002

Activation of G protein-coupled receptors by agonists involves significant movement of transmembr... more Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TM) following binding of agonist. The underlying structural mechanism by which receptor activation takes place is largely unknown but can be inferred by detecting variability within the environment of the ligand-binding pocket, which constitutes a water-accessible crevice surrounded by the seven TM helices. Using the substituted cysteine accessibility method, we initially identified those residues within the seventh transmembrane domain (TM7) of wild type angiotensin II type 1 (AT 1 ) receptor that contribute to forming the binding site pocket. We have substituted successively TM7 residues ranging from Ile 276 to Tyr 302 to cysteine. Treatment of A277C, V280C, T282C, A283C, I286C, A291C, and F301C mutant receptors with the charged sulfhydryl-specific alkylating agent MTSEA significantly inhibited ligand binding, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT 1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was greatly reduced for TM7 reporter cysteines engineered in a constitutively active mutant of the AT 1 receptor. Our data suggest that upon activation, TM7 of the AT 1 receptor goes through a pattern of helical movements that results in its distancing from the binding pocket per se. These studies support accumulating evidence whereby elements of TM7 of class A GPCRs promote activation of the receptor through structural rearrangements.

Canadian Journal of Physiology and Pharmacology, 2002

We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards t... more We have applied photoaffinity labelling methods combined with site-directed mutagenesis towards the two principal angiotensin II (AnglI) receptors AT1 and AT2 in order to determine contact points between AngII and the two receptors. We have first identified the receptor contact points between an N- and a C-terminal residue of the AngII molecule and the AT1 receptor and constructed with this stereochemical restriction a molecular model of AT1. A similar approach with a modified procedure of photoaffinity labelling has allowed us now to determine contact points also in the AT2 receptor. Molecular modelling of AT2 on the rhodopsin scaffold and energy minimisation of AngII binding into this AT2 model produced a model strikingly similar to the AT11 structure. Superposition of the experimentally obtained contact points of AngII with AT2 upon this model revealed excellent congruence between the experimental and modelling results. (i) athough AT1 and AT2 have quite low sequence homology, they both bind AngII with similar affinity and in an almost identical fashion, as if the ligand dictates the way it has to be bound, and (ii) in its bound form, AngII adopts an extended conformation in both AT1 and AT2, contrary to all previous predictions.

Biochemistry, 2002

The human angiotensin II type 1 receptor (hAT 1 ) was photolabeled with a high-affinity radiolabe... more The human angiotensin II type 1 receptor (hAT 1 ) was photolabeled with a high-affinity radiolabeled photoreactive analogue of AngII, 125 I-[Sar 1 , Val 5 , p-Benzoyl-L-phenylalanine 8 ]AngII ( 125 I-[Sar 1 ,Bpa 8 ]AngII). Chemical cleavage with CNBr produced a 7 kDa fragment (285-334) of the C-terminal portion of the hAT 1 . Manual Edman radiosequencing of photolabeled, per-acetylated, and CNBr-fragmented receptor showed that ligand incorporation occurred through Phe 293 and Asn 294 within the seventh transmembrane domain of the hAT 1 . Receptor mutants with Met introduced at the presumed contact residues, F293M and N294M, were photolabeled and then digested with CNBr. SDS-PAGE analysis of those digested mutant receptors confirmed the contact positions 293 and 294 through ligand release induced by CNBr digestion. Additional receptor mutants with Met residues introduced into the N-and C-terminal proximity of those residues 293 and 294 of the hAT 1 produced, upon photolabeling and CNBr digestion, fragmentation patterns compatible only with the above contact residues. These data indicate that the C-terminal residue of AngII interacts with residues 293 and 294 of the seventh transmembrane domain of the human AT 1 receptor. Taking into account a second receptor-ligand contact at the second extracellular loop and residue 3 of AngII (

Angiotensin Protocols, 2000

Photoaffinity labeling is a useful method to covalently bind two interacting moieties whether the... more Photoaffinity labeling is a useful method to covalently bind two interacting moieties whether they be substrate and enzyme or ligand and receptor. Irreversibly labeling any particular molecule is a practical way of detecting the latter throughout the course of a characterization or a purification procedure.

Journal of Medicinal Chemistry, 2014

We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homol... more We studied the factors affecting the selectivity of peptidomimetic inhibitors of the highly homologous proteases matriptase and matriptase-2 across subpockets using docking simulations. We observed that the farther away a subpocket is located from the catalytic site, the more pronounced its role in selectivity. As a result of our exhaustive virtual screening, we biochemically validated novel potent and selective inhibitors of both enzymes.

Pharmacogenetics and Genomics, 2010

Molecular Pharmacology, 2008

Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that a... more Class A (rhodopsin-like) G protein-coupled receptors possess conserved residues and motifs that are important for their specific activity. In the present study, we examined the role of residue Asp97(2.50) as well as residues Glu147(3.49), Arg148(3.50), and Tyr149(3.51) of the ERY motif on the functionality of the urotensin II receptor (UT). Mutations D97(2.50)A, R148(3.50)A, and R148(3.50)H abolished the ability of UT to activate phospholipase C, whereas mutations E147(3.49)A and Y149(3.51)A reduced the ability to activate PLC by 50%. None of the mutants exhibited constitutive activity. However, R148(3.50)A and R148(3.50)H promoted ERK1/2 activation, which was abolished by 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478), an inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase activity. Both these mutants were capable of directly activating EGFR, which confirmed that they activated the mitogen-activated protein kinase (MAPK) pathway by a Galpha(q/11)-independent transactivation of EGFR. The D97(2.50)A, R148(3.50)A, and R148(3.50)H mutants did not readily internalize and did not promote translocation or colocalize with beta-arrestin2-GFP. Finally, the agonist-induced internalization of the E147(3.49)A mutant receptor was significantly increased compared with wild-type receptor. This study highlights the major contribution of the conserved Asp(2.50) residue to the functionality of the UT receptor. The Arg residue in the ERY motif of UT is an important structural element in signaling crossroads that determine whether Galpha(q/11)-dependent and -independent events can occur.

Molecular Pharmacology, 2007

The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predi... more The role of transmembrane domain six (TMD6) of the angiotensin II type 1 receptor, which is predicted to undergo conformational changes after agonist binding, was investigated using the substituted-cysteine accessibility method. Each residue in the Lys240 -Leu265 fragment was mutated, one at a time, to a cysteine. The resulting mutants were expressed in COS-7 cells, which were subsequently treated with the charged sulfhydrylspecific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). This treatment led to a significant reduction in binding of 125 I-[Sar 1 ,Ile 8 ]AngII to the F249C, H256C, T260C, and V264C mutant receptors, suggesting that these residues orient themselves within the water-accessible binding pocket of the AT 1 receptor. It is noteworthy that this pattern of acquired MTSEA sensitivity was altered for TMD6 cysteines engineered in a constitutively active AT 1 receptor. Indeed, mutant F249C was insensitive to MTSEA treatment, whereas the sensitivity of mutant V264C decreased. Under these conditions, one other mutant, F261C, was found to be sensitive to MTSEA treatment. Our results suggest that constitutive activation of the AT 1 receptor causes TMD6 to pivot. This movement moves the top (extracellular side) of TMD6 toward the binding pocket and simultaneously distances the bottom (intracellular side) away from the binding pocket. Using this approach, we identified key elements within TMD6 that contribute to the activation of class A GPCRs through structural rearrangements.

The Journal of biological chemistry, 2015

Biased signalling represents the ability of G protein-coupled receptors (GPCRs) to engage distinc... more Biased signalling represents the ability of G protein-coupled receptors (GPCRs) to engage distinct pathways with various efficacies depending on the ligand used or on mutations in the receptor. The angiotensin-II type 1 receptor (AT1), a prototypical class A GPCR, can activate various effectors upon stimulation with the endogenous ligand AngII including the Gq/11 protein and β-arrestins. It is believed that the activation of those two pathways can be associated to distinct conformations of the AT1 receptor. To verify this hypothesis, microseconds of molecular dynamics simulations (MD) were computed to explore the conformational landscape sampled by the WT-AT1 receptor, the N111G-AT1 receptor (constitutively active and biased for the Gq/11 pathway) and the D74N-AT1 receptor (biased for the beta-arrestin 1 and 2 pathways) in their apo forms and in complex with AngII. The MD simulations of the AngII-WT-AT1, N111G-AT1 and AngII-N111G-AT1 receptors revealed specific structural rearrangem...

Nature Communications, 2015

Molecular pharmacology, Jan 25, 2015

The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the act... more The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein-coupled receptor. The AT1 receptor engages and activates several signalling pathways, including heterotrimeric G proteins Gq and G12, as well as the ERK1/2 pathway. Additionally, following stimulation, β-arrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favour the activation of some signalling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of 6 different signalling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4 and 8, we sough...

Journal of Peptide Science, 2007

Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protei... more Photoaffinity labelling is regularly used to investigate proteins, including peptidergic G protein-coupled receptors (GPCR). To this purpose benzophenone photolabels have been widely used to identify many contact residues in ligand-binding pockets. The three-dimensional binding environment of the human angiotensin II type 1 receptor hAT 1 has been determined using an iterative methionine mutagenesis strategy based on the photochemical properties and preferential incorporation of benzophenone onto methionine. This has led to the construction of a ligand-bound receptor structure. The present study investigated the effect of temperature on the accessibility of some of these contact points. The hAT 1 receptor and two representative Met mutants (H256M-hAT 1 and F293M-hAT 1 ) from the iterative mutagenesis study were photolabelled with the benzophenoneligand 125 I-[Sar 1 , Bpa 8 ]AngII at temperatures ranging from −15°C to 37°C. Labelled receptors were partially purified and digested with cyanogen bromide to identify the contact points or segments. There were no changes in receptor contacts or labelling in the 7th transmembrane domains (TMD) of hAT 1 and F293M-hAT 1 across the temperature range. However, a temperature-dependent change in the ligand-receptor contact of H256M-hAT 1 was observed. At −15°C, H256M labelling was identical to that of hAT 1 , indicating that the interaction was specific to the 7th TMD. Significant labelling changes were observed at higher temperatures and at 37°C labelling occurred almost exclusively at mutated residue H256M-hAT 1 in the 6th TMD. Simultaneous competitive labelling of different areas of this target protein indicated that the ligand-receptor structure became increasingly fluctual at physiological temperatures, while a more compact, low mobility, and low energy conformation prevailed at low temperatures.

Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 p... more Extracts from BSC-40 cells infected with vaccinia recombinants expressing either the yeast KEX2 prohormone endoprotease or a human structural homologue (fur gene produc0 contained an elevated level of a membrane-associated endoproteolytic activ- ity that could cleave at pairs of basic amino acids (-LysArg- and -ArgArg-). The fur-directed activity (fu- fin) shared many properties with Kex2p including ac- tivity at pH

The International Journal of Biochemistry & Cell Biology, 2009

a b s t r a c t ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in... more a b s t r a c t ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR 46 , RRR 69 and RRRRR 261 ) suggested that proADAMTS5 processing occurs after Arg 261 . That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

PloS one, 2014

Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine prot... more Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors. To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P') sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 indi...

European Journal of Biochemistry, 1997

Production of endothelin-1 is thought to be a three-step process consisting of an initial proteol... more Production of endothelin-1 is thought to be a three-step process consisting of an initial proteolytic cleavage of the proendothelin-1 precursor to big endothelin-I-Lys-Arg, C-terminal trimming by a carboxypeptidase and further processing of the big endothelin-I peptide to endothelin-1 by endothelinconverting enzyme (ECE). To further clarify the mechanism of processing in the biosynthesis of endothelin-1, we introduced a point mutation into endothelin-I cDNA to replace the Arg in the -4 position of the recognition motifs of furin-like convertase in human preproendothelin-1 (Arg49 or Arg89) by Gly. When mutant cDNAs were expressed in Chinese hamster ovary (CH0)-K1 cells, they failed to be processed at the mutated processing signal, suggesting that the Arg-Ser-Lys-Arg motifs of preproendothelin-1 are recognized by CHO-K1 furin-like convertase. Co-transfection with ECE-1 cDNA revealed that cleavage at ArgS2 is not essential for cleavage by ECE-1, but that cleavage at Arg92 is critical. Although a high-molecular-mass form of endothelin-1 is produced by processing by ECE-1 without cleavage at Arg52, it did not evoke Ca2+ transient in endothelin,-receptor-expressing cells. In conclusion, prior cleavage at Arg92 by furin-like convertase is absolutely necessary for cleavage by ECE-1 at Trp73 to produce mature endothelin-1 .

Prostaglandins, Leukotrienes and Essential Fatty Acids, 1998

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory ... more Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases. NSAIDs inhibit cyclooxygenase (COX), the rate limiting enzyme responsible for the conversion of arachidonic acid into prostaglandins. Recent studies have shown the existence of two isoforms of cyclooxygenase: COX-l, now often referred to as the constitutive form, and COX-2, an inducible form which is the major isoenzyme involved in prostaglandin synthesis in inflammation and other pathological situations. Since inhibition of prostaglandin production in tissues where they play a physiological role leads to important side effects, a COX-2 preferential inhibitor would present therapeutical advantages. In the present study, we evaluated the inhibitory properties of cyclooxygenase inhibitors on human COX-1 and COX-2 using a heterologous expression system. We investigated instantaneous inhibition and pre-incubation inhibition as well as time recovery of cyclooxygenase activity assays with the aid of four NSAIDs: mefenamic acid, indomethacin, aspirin and NS-398. Our results demonstrate that instantaneous inhibition assays have little correlation with clinical results. Inhibition assays using pre-incubation with the drugs tested, however, more closely resemble the data from in vivo studies. Cyclooxygenase recovery assays enabled better characterization of simple competitive inhibitors, competitive reversible time-dependent inhibitors and irreversible time-dependent inhibitors. The data illustrate the usefulness of our system in allowing a better determination of the pharmacological characteristics of NSAIDs as well as permitting a comparison among different drugs.

Proceedings of the National Academy of Sciences, 1991

Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrineprecursor... more Two mammalian gene products, PC2 and PC3, have been proposed as candidate neuroendocrineprecursor processing enzymes based on the structural similarity of their catalytic domains to that of the yeast precursorprocessing endoprotease Kex2. In this report we demonstrate that these two proteases can cleave proopiomelanocortin (POMC) in the secretory pathway of mammalian cells. Similarly to pituitary corticotrophs, PC3 expressed in processingdeficient BSC-40 cells cleaved native mouse POMC at the -Lys-Argsites flanking corticotropin. The -Lys-Argwithin 13-lipotropin was less efficiently cleaved to release f-endorphin.

Pharmacogenetics and Genomics, 2010