Cell-surface processing of pro-ADAMTS9 by furin.

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium.

Role of N-glycan-dependent quality control in the cell-surface expression of the AT1 receptor.

Most G protein-coupled receptors (GPCRs) are N-glycosylated proteins but the role of this post-translational modification in GPCR biosynthesis has not been extensively studied. We previously showed that the non-glycosylated AT(1) receptor is inefficiently expressed at the cell surface. In this study, we addressed whether AT(1) interacts with elements of the ER-based quality control processes.

Mass spectrometric peptide fingerprinting of proteins after Western blotting on polyvinylidene fluoride and enhanced chemiluminescence detection.

The combined use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry has become a powerful and widely used tool in proteome studies. Following separation by electrophoresis, proteins can be transferred to an inert support such as polyvinylidene fluoride (PVDF) or nitrocellulose (NC) for the visualization of individual or specific classes of proteins by immunochemical detection methods. We developed a method that allows the mass spectrometric analysis of peptides derived from proteins detected by Western blotting on PVDF.

New and automated MSn approaches for top-down identification of modified proteins.

An automated top-down approach including data-dependent MS(3) experiment for protein identification/characterization is described. A mixture of wild-type yeast proteins has been separated on-line using reverse-phase liquid chromatography and introduced into a hybrid linear ion trap (LTQ) Fourier transform ion cylclotron resonance (FTICR) mass spectrometer, where the most abundant molecular ions were automatically isolated and fragmented. The MS(2) spectra were interpreted by an automated algorithm and the resulting fragment mass values were uploaded to the ProSight PTM search engine to identify three yeast proteins, two of which were found to be modified.

The constitutively active N111G-AT1 receptor for angiotensin II modifies the morphology and cytoskeletal organization of HEK-293 cells.

The expression of a constitutively active G protein-coupled receptor is expected to trigger diverse cellular changes ranging from normal to adaptive responses. We report that confluent HEK-293 cells stably expressing the constitutively active mutant N111G-AT1 receptor for angiotensin II spontaneously exhibited dramatic morphological changes and cytoskeletal reorganization. Phase-contrast microscopy revealed that these cells formed a dense monolayer, whereas cells expressing the WT-AT1 receptor displayed large intercellular spaces and numerous filopodia.

Determining the environment of the ligand binding pocket of the human angiotensin II type I (hAT1) receptor using the methionine proximity assay.

The peptide hormone angiotensin II (AngII) binds to the AT0 (angiotensin type 1) receptor within the transmembrane domains in an extended conformation, and its C-terminal residue interacts with transmembrane domain VII at Phe-293/Asn-294. The molecular environment of this binding pocket remains to be elucidated. The preferential binding of benzophenone photolabels to methionine residues in the target structure has enabled us to design an experimental approach called the methionine proximity assay, which is based on systematic mutagenesis and photolabeling to determine the molecular environment of this binding pocket.

Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor.

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT(1) receptor at the cell surface.

Stability of mutant serpin/furin complexes: dependence on pH and regulation at the deacylation step.

Furin proteolytically cleaves a wide variety of proprotein substrates mainly within the trans-Golgi network (TGN) but also at the cell membrane and in endosomal compartments where pH is more acidic. Incorporation of furin recognition sequences within the reactive site loop (RSL) of alpha(1)-antitrypsin (AT) leads to the production of furin inhibitors. In an attempt to design more stable, potent, and specific serpin-based inhibitors, we constructed a series of AT and alpha(1)-antichymotrypsin (ACT) mutants by modifying the P(7)-P(1) region of their RSLs.

ESTIMA, a tool for EST management in a multi-project environment.

Background: Single-pass, partial sequencing of complementary DNA (cDNA) libraries generates thousands of chromatograms that are processed into high quality expressed sequence tags (ESTs), and then assembled into contigs representative of putative genes. Usually, to be of value, ESTs and contigs must be associated with meaningful annotations, and made available to end-users.

Results: A web application, Expressed Sequence Tag Information Management and Annotation (ESTIMA), has been created to meet the EST annotation and data management requirements of multiple high-throughput EST sequencing projects.

Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization.

Most G-protein-coupled receptors that undergo agonist-dependent internalization require the presence of specific cytoplasmic-tail residues to initiate interactions with proteins of the endocytic machinery. Here we show that the UT receptor (urotensin II receptor) undergoes internalization, and that specific serine residues of the receptor's cytoplasmic tail participate in this process. We first observed a time-dependent increase in internalization of the UT receptor expressed in COS-7 cells following binding of the agonist urotensin II.

Analysis of the third transmembrane domain of the human type 1 angiotensin II receptor by cysteine scanning mutagenesis.

Activation of G protein-coupled receptors by agonists involves significant movement of transmembrane domains (TMD) following agonist binding. 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 is a water-accessible crevice surrounded by the seven TMD helices. Using the substituted-cysteine accessibility method, we identified the residues within the third TMD of the wild-type angiotensin II (AT1) receptor that contribute to the formation of the binding site pocket.

Down-regulation of inositol 1,4,5-trisphosphate receptor in cells stably expressing the constitutively active angiotensin II N111G-AT(1) receptor.

The diverse cellular changes brought about by the expression of a constitutively active receptor are poorly understood. QBI-human embryonic kidney 293A cells stably expressing the constitutively active N111G-AT(1) receptor (N111G cells) showed elevated levels of inositol phosphates and frequent spontaneous intracellular Ca(2+) oscillations. Interestingly, Ca(2+) transients triggered with maximal doses of angiotensin II were much weaker in N111G cells than in wild-type cells.

ProSight PTM: an integrated environment for protein identification and characterization by top-down mass spectrometry.

ProSight PTM (https://prosightptm.scs.uiuc.

ADAMTS7B, the full-length product of the ADAMTS7 gene, is a chondroitin sulfate proteoglycan containing a mucin domain.

We have characterized ADAMTS7B, the authentic full-length protein product of the ADAMTS7 gene. ADAMTS7B has a domain organization similar to that of ADAMTS12, with a total of eight thrombospondin type 1 repeats in its ancillary domain. Of these, seven are arranged in two distinct clusters that are separated by a mucin domain.

Identification of prodomain determinants involved in ADAMTS-1 biosynthesis.

The metalloprotease ADAMTS-1 (a disintegrin and metalloprotease with thrombospondin type I motif), similarly to other members of the ADAMTS family, is initially synthesized as a zymogen, proADAMTS-1, that undergoes proteolytic processing at the prodomain/catalytic domain junction by serine proteinases of the furin-like family of proprotein convertases. The goals of this study were to identify residues of the prodomain that play an essential role in ADAMTS-1 processing and to determine the identity of the convertase required for zymogen processing. To gain insight into the putative roles of specific prodomain residues in ADAMTS-1 biosynthesis, we performed biosynthetic labeling experiments in transiently transfected human embryonic kidney 293 cells expressing wild-type and prodomain mutants of proADAMTS-1.

Identification of furin pro-region determinants involved in folding and activation.

The pro-region of the subtilisin-like convertase furin acts early in the biosynthetic pathway as an intramolecular chaperone to enable proper folding of the zymogen, and later on as an inhibitor to constrain the activity of the enzyme until it reaches the trans -Golgi network. To identify residues that are important for pro-region function, we initially identified amino acids that are conserved among the pro-regions of various mammalian convertases. Site-directed mutagenesis of 17 selected amino acids within the 89-residue pro-region and biosynthetic labelling revealed that I60A-furin and H66A-furin were rapidly degraded in a proteasome-dependent manner, while W34A-furin and F67A-furin did not show any autocatalytic activation.

The constitutively active N111G-AT1 receptor for angiotensin II maintains a high affinity conformation despite being uncoupled from its cognate G protein Gq/11alpha.

Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor.

Constitutive activation of the angiotensin II type 1 receptor alters the spatial proximity of transmembrane 7 to the ligand-binding pocket.

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 (AT1) receptor that contribute to forming the binding site pocket.

Characterization of ADAMTS-9 and ADAMTS-20 as a distinct ADAMTS subfamily related to Caenorhabditis elegans GON-1.

We demonstrate that in humans, two metalloproteases, ADAMTS-9 (1935 amino acids) and ADAMTS-20 (1911 amino acids) are orthologs of GON-1, an ADAMTS protease required for gonadal morphogenesis in Caenorhabditis elegans. ADAMTS-9 and ADAMTS-20 have an identical modular structure, are distinct in possessing 15 TSRs and a unique C-terminal domain, and have a similar gene structure, suggesting that they comprise a new subfamily of human ADAMTS proteases. ADAMTS20 is very sparingly expressed, although it is detectable in epithelial cells of the breast and lung.

Methionine proximity assay, a novel method for exploring peptide ligand-receptor interaction.

Probing G-protein coupled receptor (GPCR) structures is a priority in the functional and structural understanding of GPCRs. In the past, we have used several approaches around photoaffinity labeling in order to establish contact points between peptide ligands and their cognate receptors. Such contact points are helpful to build reality based molecular models of GPCRs and to elucidate their activation mechanisms.

Photolabelling the rat urotensin II/GPR14 receptor identifies a ligand-binding site in the fourth transmembrane domain.

A urotensin II (U-II) peptide analogue containing the photoreactive p -benzoyl-L-phenylalanine (Bz-Phe) in the sixth position was used to identify ligand-binding sites of the rat U-II receptor, also known as GPR14. [Bz-Phe(6)]U-II bound the receptor expressed in COS-7 cells with high affinity (IC(50) 0.7 nM) and was as potent as U-II in the agonist-induced production of inositol phosphate.

Residues 293 and 294 are ligand contact points of the human angiotensin type 1 receptor.

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).

A polyaromatic caveolin-binding-like motif in the cytoplasmic tail of the type 1 receptor for angiotensin II plays an important role in receptor trafficking and signaling.

The type 1 receptor for angiotensin II (AT(1)) is a member of the G protein-coupled receptor family. The presence of a caveolin-binding-like motif (phiXphiXXXXphiXXphi where phi is an aromatic residue) within the cytoplasmic tail of the AT(1) receptor suggests an implication for caveolae in the functionality of this receptor. We constructed a mutant AT(1) receptor where each of the aromatic residues in the caveolin-binding-like motif were replaced by alanine (AT(1)-YFFY/A).

Polyethylene glycol conjugation at Cys232 prolongs the half-life of alpha1 proteinase inhibitor.

alpha1 Proteinase inhibitor (alpha1PI), a natural inhibitor of the serine proteinase leukocyte elastase, is also an intravenous therapeutic agent used to treat hereditary emphysema and may be useful in other respiratory disorders. However, to achieve sustained suppression of leukocyte elastase, alpha1PI must be given frequently and in large amounts, thus limiting its clinical use. We hypothesized that conjugating alpha1PI with polyethylene glycol (PEG) at Cys(232) could extend the in vivo half-life of alpha1PI in blood and lung.

Ectodomain shedding of furin: kinetics and role of the cysteine-rich region.

Furin, a member of the subtilisin-like pro-protein convertase family, is a type I membrane protein that undergoes ectodomain shedding. Metabolic labeling of cells stably expressing furin demonstrated that the shed form of furin is detected after 30 min. Moreover, sequence analysis revealed that specific residues of the cysteine-rich region of furin aligned with those of tumor necrosis factor receptor, which is also shed.