We have characterized ADAMTS7B, the authentic fulllength 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. Unique to the ADAMTS family, ADAMTS7B is modified by attachment of the glycosaminoglycan chondroitin sulfate within the mucin domain, thus rendering it a proteoglycan. Glycosaminoglycan addition has potentially important implications for ADAMTS7B cellular localization and for substrate recognition. Although not an integral membrane protein, ADAMTS7B is retained near the cell surface of HEK293F cells via interactions involving both the ancillary domain and the prodomain. ADAMTS7B undergoes removal of the prodomain by a multistep furin-dependent mechanism. At least part of the final processing event, i.e. cleavage following Arg 220 (mouse sequence annotation), occurs at the cell surface. ADAMTS7B is an active metalloproteinase as shown by its ability to cleave ␣ 2 -macroglobulin, but it does not cleave specific peptide bonds in versican and aggrecan attacked by ADAMTS proteases. Together with ADAMTS12, whose primary structure also predicts a mucin domain, ADAMTS7B constitutes a unique subgroup of the ADAMTS family. The extracellular matrix (ECM)1 is an information-rich assembly influencing cell proliferation, apoptosis, and cell migration. Proteases have an essential role in modulating the environmental cues that ECM provides for tissue morphogenesis, homeostasis, and disease progression. Metalloproteases, especially matrix metalloproteases, have a conspicuous role in ECM degradation as well as in proteolysis of cell-surface and soluble proteins (1, 2). Another metalloprotease family, ADAM (a disintegrin and metalloprotease domain), contains transmembrane enzymes with a major role in ectodomain shedding of cell-surface molecules, but a negligible function in ECM proteolysis (3). The active site of ADAM proteases, unlike that of the matrix metalloproteases, is of the reprolysin (snake venom zinc metalloprotease) type (3). The recent discovery of the ADAMTS (a disintegrin-like and metalloprotease domain with thrombospondin type 1 motif) family brought to light metalloproteases that contain a reprolysintype catalytic site, but, unlike the ADAM proteases, are secreted enzymes with a prominent role in ECM proteolysis.ADAMTS proteases have a characteristic modular structure whose hallmark is the presence of one or more thrombospondin type 1 repeats (TSRs) (4). In the short period of time since the discovery of ADAMTS1 in 1997 (4), important functions have been attributed to a number of family members, and mutations of two of these enzymes have been shown to cause human genetic disorders. Inactivating mutations of ADAMTS13 cause inherited thrombocytopenic purpura due to a failure to process von Willebrand factor (5). Mutations of ADAMTS2, a procollagen aminopropeptidase, cause skin fragility in a var...

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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. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furindeficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cellsurface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.Zinc metalloendopeptidases, like most proteases, are synthesized as zymogens, and the N-terminal propeptide is usually excised. Propeptide excision usually leads to enzymatic activation, an important regulatory event, and it can occur intracellularly, at the cell surface, or extracellularly through a variety of proteolytic mechanisms. In one such mechanism, the propeptide is proteolytically excised by serine proteases of the mammalian subtilisin-like proprotein convertase (PC) 4 family (1-5). This mechanism is used by some MMPs (6, 7), many ADAMs (8 -10), and all ADAMTS proteases studied thus far (11,12). In these proteases, removal of the propeptide appears to be mediated by the most widely distributed PC, furin, and occurs within the constitutive secretory pathway, specifically in the trans-Golgi network (TGN) (7-9, 13, 14).Furin is the best studied of the seven PCs implicated in proprotein processing within the constitutive secretory pathway, and it is present in virtually all cells (1, 15). It is a type I transmembrane protein that is itself synthesized as a zymogen that undergoes autocatalytic intramolecular activation (16). Furin cleaves on the carboxyl side of a consensus recognition site that is rich in basic residues (e.g. Arg-Xaa-Arg/Lys-Arg2) (2,4,5,17). Most furin resides in the TGN, but some is present at the plasma membrane and shuttles between the cell surface and the . Furin is also shed from cells and may be functional in the extracellular space (21). Microbial toxins such as the anthrax protective antigen and diphtheria toxin are processed by cell-surface furin, with important implications for their toxicity (20,22). However, the physiological role of cell-surface or secreted furin in processing endogenous cellular products has remained elusive.The ADAMTS proteases are a family of 19 secreted enzymes, of which some have critical physiological functions and have been implicated in inherited human disorders, namely Ehlers-Danlos syndrome type VIIC (ADAMTS2), Weill-Marchesani syndrom...

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg 74 2 and Arg 287 2) the ADAMTS9 propeptide was also furin-processed at Arg 209 . Substitution of Ala for Arg 74 , Arg 209 , and Arg 287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface. ADAMTS4 proteases have critical roles in many biological processes and in inherited and acquired human disorders (1-5). The 19 enzymes of this family share a conserved organization comprising an N-terminal metalloprotease domain and a C-terminal ancillary domain, which contains the thrombospondin type-1 repeats that are the hallmark of the family (6). ADAMTS9 is the largest enzyme of the family, containing 15 thrombospondin type-1 repeats, and its mRNA is widely expressed during embryonic development and in adult tissues (7-9). In previously published work, we showed that when expressed in COS-1 or HEK293F cells, ADAMTS9 is located at the cell surface or within the pericellular matrix (8), suggesting that, despite the lack of a membrane anchor, it could be considered as an operational cell-surface protease. ADAMTS9 can cleave the large aggregating proteoglycans aggrecan and versican (8), suggesting a role in turnover of extracellular matrix. Thus, like its Caenorhabditis elegans ortholog, Gon-1, which is required for cell migration during gonadal morphogenesis (10), it is possible that ADAMTS9 participates in extracellular proteolysi...

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β1-adrenergic receptor (β1AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to Gz and G12 pathways. The signalling cascade linking the β1AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both Gs and G12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.