Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.

Marriott, David; Gillece-Castro, Beth; Gorman, Cornelia M.

doi:10.1210/mend.6.9.1435788

Cited by 23 publications

(15 citation statements)

References 28 publications

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“…These phage were bound to hGHbp immobilized on microtiter plates and treated with -300 nM furin at 37 "C for 2 h. Approximately 4 times as many phage were released when ATMD-furin was added compared to a control experiment with buffer alone. This enrichment was less than the IO-100-fold enrichments observed in previous sub-CMV enhancer not cleaved by furin (Marriott et al, 1992). In either case, no promoter more phage were released on incubation with furin than with buffer alone.…”

Section: Substrate Phage Selectionmentioning

confidence: 56%

“…1) were initially performed with phage containing a known furin substrate sequence (RKKRIQ: Marriott et al, 1992) that was inserted between the hGH variant and the truncated gene 111 protein. These phage were bound to hGHbp immobilized on microtiter plates and treated with -300 nM furin at 37 "C for 2 h. Approximately 4 times as many phage were released when ATMD-furin was added compared to a control experiment with buffer alone.…”

Section: Substrate Phage Selectionmentioning

confidence: 99%

“…A vector containing the human cytomegalovirus enhancer and promoter followed by an intron and containing an SV40 polyadenylation signal (Marriott et al, 1992) was digested with Xba I and EcoR I and a 4.7-kb fragment was isolated. The same vector containing the full-length cDNA sequence of mouse furin was digested with Xba I and Pst I, and a 2.13-kb fragment was isolated that lacked the last 267 bp, encoding the transmembrane and cytoplasmic domains.…”

Section: Construction Of a Secreted Form Of Furin (Atmd-furin)mentioning

confidence: 99%

“…Following electrophoresis, the proteins were electroblotted onto a PVDF membrane, blocked with 2% BSA in TBST buffer (10 mM Tris-HC1, Construction of furin substrate phage, PC1 substrate phage, and substrate phage libraries DNA sequences coding for known furin and PC1 substrates from human relaxin (Marriott et al, 1992) were introduced between a variant hGH gene and truncated gene 111 in the phagemid vector pDM0612 (Matthews & Wells, 1993) by cassette mutagenesis. The hGH variant contains the mutations R167N, D171N, K172S, F176Y, I178T, FlOA, M14W, H18D, and H21N, and has been shown to bind very tightly to the hGHbp (Lowman et al, 1991).…”

Section: Immunoblot Analysismentioning

confidence: 99%

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A survey of furin substrate specificity using substrate phage display

Goodman²,

et al. 1994

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The substrate specificity of furin, a mammalian enzyme involved in the cleavage of many constitutively expressed protein precursors, was studied using substrate phage display. In this method, a multitude of substrate sequences are displayed as fusion proteins on filamentous phage particles and ones that are cleaved can be purified by affinity chromatography. The cleaved phage are propagated and submitted to additional rounds of protease selection to further enrich for good substrates. DNA sequencing of the cleaved phage is used to identify the substrate sequence. After 6 rounds of sorting a substrate phage library comprising 5 randomized amino acids (xxxxx), virtually all clones had an RxxR motif and many had Lys, Arg, or Pro before the second Arg. Nine of the selected sequences were assayed using a substrate-alkaline phosphatase fusion protein system. All were cleaved after the RxxR, and some substrates with Pro or Thr in P2 were also found to be cleaved as efficiently as RxKR or RxRR. To further elaborate surrounding determinants, we constructed 2 secondary libraries (xxRx(K/R)Rx and xxRxPRx). Although no consensus developed for the latter library, many of the sequences in the the former library had the 7-residue motif (L/P)RRF(K/R)RP, suggesting that the furin recognition sequence may extend over more than 4 residues. These studies further clarify the substrate specificity of furin and suggest the substrate phage method may be useful for identifying consensus substrate motifs in other protein processing enzymes.Keywords: furin; substrate phage display; substrate specificity; subtilisin-like protease Site-specific proteolysis is one of the most widespread and important posttranslational modifications. Many proteins, including neuropeptides, peptide hormones, enzymes, and viral glycoproteins, are synthesized as inactive precursors that require specific endoproteolytic cleavage for maturation. Recently, a novel family of mammalian subtilisin-like proteases has been discovered that appears to be involved in intracellular processing of spe- cific protein precursors (for recent reviews, see Steiner et al., 1992, andSmeekens, 1993).It is useful to establish which precursor is processed by which enzyme to better understand the regulation of processing in vivo and to design potential therapeutics for affecting a particular processing step. Linking a precursor with a processing enzyme is often done by showing that they are expressed in the same tissues, that they co-localize at the same time and place within the cell, and that the precursor has a sequence that can be cut by the enzyme. This latter aspect is facilitated by understanding the substrate specificity of the enzyme, which can be very time consuming because it traditionally involves the synthesis and kinetic analysis of many peptides or modified protein precursors.Recently, a technique called substrate phage display has been developed (Matthews &Wells, 1993), which allows one to rapidly survey the specificity of a protease using an iterative selection ...

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Section: Substrate Phage Selectionmentioning

confidence: 56%

Section: Substrate Phage Selectionmentioning

confidence: 99%

Section: Construction Of a Secreted Form Of Furin (Atmd-furin)mentioning

confidence: 99%

Section: Immunoblot Analysismentioning

confidence: 99%

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A survey of furin substrate specificity using substrate phage display

Goodman²,

et al. 1994

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“…To allow efficient cleavage of the prepropeptide during post-translational modifications and the secretion of mature peptides, HEK293T cells were routinely cotransfected with the select expression construct and a one-tenth aliquot of a convertase expression vector (42). Cells were cultured in Dulbecco's modified Eagle's medium/F-12 media supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 g/ml streptomycin in a water-saturated atmosphere containing 5% CO 2 at 37°C.…”

Section: Subcloning Of Wild Type Human Relaxin Family Genes Andmentioning

confidence: 99%

Regulation of Receptor Signaling by Relaxin A Chain Motifs

Park

Semyonov

et al. 2008

Journal of Biological Chemistry

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Relaxin peptides are important hormones for the regulation of reproductive tissue remodeling and the renal cardiovascular system during pregnancy. Recent studies demonstrated that two of the seven human relaxin family peptides, relaxin H2 (RLN2) and INSL3, signal exclusively through leucine-rich repeat-containing G protein-coupled receptors, LGR7 and LGR8. Although it was well characterized that an RXXXRXXI motif at the RLN2 B chain confers receptor activation activity, it is not clear what roles RLN2 A chain plays in receptor interaction. Analyses of relaxin family genes on syntenic regions of model tetrapods showed that the A chain of RLN2 orthologs exhibited a greater sequence divergence as compared with the receptorbinding domain-containing B chain, foreshadowing a potential role in receptor interactions; hence, defining receptor selectivity in this fast evolving peptide hormone. To test our hypothesis that select residues in the human RLN2 A chain play key roles in receptor interaction, we studied mutant peptides with residue substitution(s) in the A chain. Here, we showed that alanine substitution at the A16 and A17 positions enhances LGR8-activation activity of RLN2, whereas mutation at the A22-23 region (RLN2 A22-23 ) ablates LGR8, but not LGR7, activation activity. In addition, we demonstrated that the functional characteristics of the RLN2 A22-23 mutant are mainly attributed to modifications at the Phe A23 position. Taken together, our studies indicated that Thr A16 , Lys A17 , and Phe A23 constitute part of the receptorbinding interface of human RLN2, and that modification of these residues has led to the generation of novel human RLN2 analogs that would allow selective activation of human LGR7, but not LGR8, in vivo.In humans, the relaxin gene family contains a total of seven members: relaxin H1 (RLN1), 3 relaxin H2 (RLN2), relaxin 3/INSL7 (RLN3), INSL3/RLF, INSL4/EPIL, INSL5/RIF2, and INSL6/RIF1 (1-5). Among these family members, RLN2 and INSL3 signal through two leucine-rich repeat-containing GPCRs, LGR7 (RFXR1) and/or LGR8 (RFXR2). Whereas RLN2 is capable of activating both LGR7 and LGR8, INSL3 is a selective ligand for LGR8. In addition to the better characterized RLN2 and INSL3, RLN3 was shown to activate LGR7, GPCR135, and GPCR142, but not LGR8 (6 -10); therefore, the relaxin family peptides exhibit overlapping specificity on the activation of LGR7 and LGR8. Although the physiological roles of RLN3 and other family peptides remain to be studied, analysis of null mice models showed that the ablation of relaxin and INSL3 genes caused phenotypes similar to that of LGR7-and LGR8-deficient mice, suggesting that LGR7 and LGR8 are the main receptors for the single relaxin and INSL3 peptides in rodents, respectively (11-15).In addition to the role in remodeling of reproductive tissues, relaxin has been shown to effect endometrial differentiation during embryo implantation, nipple and mammary gland development, angiogenesis, wound healing, and renal cardiovascular responses (12, 13, 16 -19). Furthermore...

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Relaxin: a pregnancy hormone as central player of body fluid and circulation homeostasis

Dschietzig

Stangl

2003

Cellular and Molecular Life Sciences (CMLS)

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The peptide relaxin has long been regarded as an important hormone of pregnancy, contributing to changes in connective tissue composition as well as to regulation of implantation, myometrial activity and labor. On the other hand, the astonishing pleiotropy of this hormone escaped scientific awareness. This review focuses on new facets of relaxin, including its antifibrotic effects, its role in the control of pituitary hormone release, its vasodilator and pro-angiogenic properties and its versatile myocardial actions. Recent progress in understanding relaxin's receptor and signaling mechanisms is also highlighted. The peptide will be characterized as potential regulator of body fluid and circulation homeostasis.

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Prohormone convertase-1 will process prorelaxin, a member of the insulin family of hormones.

Cited by 23 publications

References 28 publications

A survey of furin substrate specificity using substrate phage display

A survey of furin substrate specificity using substrate phage display

Regulation of Receptor Signaling by Relaxin A Chain Motifs

Relaxin: a pregnancy hormone as central player of body fluid and circulation homeostasis

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