Solid-Phase Synthesis of Europium-Labeled Human INSL3 as a Novel Probe for the Study of Ligand−Receptor Interactions

Shabanpoor, Fazel; Hughes, Richard A.; Bathgate, Ross A. D.; Zhang, Suode; Scanlon, Denis B.; Lin, Feng; Hossain, Mohammed Akhter; Separovic, Frances; Wade, John D.

doi:10.1021/bc800127p

Cited by 56 publications

(59 citation statements)

References 38 publications

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“…This method optimizes the binding of radioactive ligand at 4°C, before restoring buffer to 37°C, a temperature permissive of receptor internalization. Total numbers of cell surface receptors have previously been measured for the RXFP1, RXFP2, and AT 1 R stable and transfected cell lines (41,47). Saturation binding studies and Scatchard analysis determined that the level of receptor expression was between 40,000 and 80,000 receptors/cell for each receptor in both stably and transiently transfected cells.…”

Section: Resultsmentioning

confidence: 99%

Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment

Callander¹,

Thomas²,

Bathgate³

2009

American Journal of Physiology-Cell Physiology

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laxin induces sustained physiological responses, which brings into question the deactivation processes typical of most G protein-coupled receptors (GPCR) for its receptor, relaxin family peptide receptor 1 (RXFP1). Here, we examined relaxin-dependent phosphorylation of RXFP1 and the related insulin-like peptide 3 (INSL3) receptor, RXFP2, as well as the capacity of these receptors to recruit ␤-arrestins and internalize in response to ligand stimulation. We confirmed in human embryonic kidney (HEK)-293T cells, expressing RXFP1 or RXFP2, that both receptors elicit prolonged cAMP responses up to 6 h after stimulation. Receptors immunoprecipitated from 32 P metabolically labeled cells were used to investigate the agonist-specific phosphorylation. Rapid and robust receptor phosphorylation was not observed for either RXFP1 or RXFP2, although some 32 P-incorporation was observed at 30 min; however, this was not statistically significant. In accord with this result, RXFP1 and RXFP2 demonstrated poor internalization in response to relaxin or INSL3, as compared with the angiotensin II type 1 receptor (AT1R), which undergoes rapid and robust phosphorylation and internalization in response to angiotensin II. Additionally, coexpression of GPCR kinases has no effect on the rate of internalization for either RXFP1 or RXFP2. Confocal microscopy was used to follow the trafficking of green fluorescent proteinlabeled ␤-arrestins after receptor activation. Neither RXFP1 nor RXFP2 activation results in recruitment of ␤-arrestins to the cell surface, whereas AT1R rapidly recruits both ␤-arrestins-1 and -2. The apparent lack of classical regulation for RXFP1 and RXFP2 provides the molecular basis for the prolonged signaling and physiological actions of relaxin and related peptides.G protein-coupled receptor; relaxin; insulin-like peptide 3 RELAXIN IS A MEMBER of the insulin-relaxin peptide family, which in humans includes 10 genes: INS, IGF1, IGF2, RLN1, RLN2, RLN3, INSL3, INSL4, INSL5, and INSL6. The human RLN2 gene encodes the relaxin peptide, which is best known for its important actions during pregnancy, including decidualization of stromal cells during implantation; growth and development of the uterus, mammary gland, and nipple; inhibition of myometrial contractions; mediation of pregnancyrelated cardiovascular changes; and regulation of plasma osmolality (reviewed in Ref. 4). Relaxin also regulates extracellular matrix turnover in the heart, lung, kidney and liver; promotion of wound healing; and counteraction of allergic reactions (reviewed in Ref. 4).The receptor for relaxin, relaxin family peptide 1 receptor (RXFP1), was identified in 2002 in conjunction with the receptor for insulin-like peptide 3 (INSL3), termed RXFP2 (20). RXFP1 and RXFP2 belong to the glycoprotein hormone receptor cluster of the ␦-group of G protein-coupled receptors (GPCR), which includes the classic glycoprotein hormone receptors follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR), and thyroid-stimulating hormone receptor (...

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Section: Resultsmentioning

confidence: 99%

Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment

Callander¹,

Thomas²,

Bathgate³

2009

American Journal of Physiology-Cell Physiology

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“…Recombinant human (rh) BMP2, BMP4, BMP6, and BMP7 were purchased from R&D Systems. Human INSL3 was prepared by optimized solid-phase synthesis of the separate A-and B-chains (26 and 31 residues, respectively), followed by sequential formation of each of the three disulfide bonds via regioselective chemical formation (50). The peptide was comprehensively chemically characterized by analytical RP-HPLC and MALDI-TOF MS to confirm its high purity.…”

Section: Methodsmentioning

confidence: 99%

Functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

Glister

Satchell

Bathgate

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Bone morphogenetic proteins (BMPs) are firmly implicated as intraovarian regulators of follicle development and steroidogenesis. Here we report a microarray analysis showing that treatment of cultured bovine theca cells (TC) with BMP6 significantly (>twofold; P < 0.01) up-or down-regulated expression of 445 genes. Insulin-like peptide 3 (INSL3) was the most heavily down-regulated gene (−43-fold) with cytochrome P450, subfamily XVII (CYP17A1) and other key steroidogenic transcripts including steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11, subfamily A1 (CYP11A1) and 3 beta-hydroxysteroid dehydrogenase type 1 (HSD3B1) also downregulated. BMP6 also reduced expression of nuclear receptor subfamily 5A1 (NR5A1) known to target the promoter regions of the aforementioned genes. Real-time PCR confirmed these findings and also revealed a marked reduction in expression of INSL3 receptor, relaxin/insulin-like family peptide receptor 2 (RXFP2). Secretion of INSL3 protein and androstenedione were also suppressed suggesting a functional link between BMP and INSL3 pathways in controlling androgen synthesis. RNAi-mediated knockdown of INSL3 reduced INSL3 mRNA (75%) and protein (94%) level and elicited a 77% reduction in CYP17A1 mRNA and 83% reduction in androstenedione secretion. Knockdown of RXFP2 also reduced CYP17A1 expression (81%) and androstenedione secretion (88%). Conversely, treatment with exogenous (human) INSL3 increased androstenedione secretion ∼twofold. The CYP17A1 inhibitor abiraterone abolished androgen secretion and reduced expression of both INSL3 and RXFP2. Collectively, these findings indicate a positive autoregulatory role for INSL3 signaling in maintaining thecal androgen production, and visa versa. Moreover, BMP6-induced suppression of thecal androgen synthesis may be mediated, at least in part, by reduced INSL3-RXFP2 signaling.growth factor | ovary | reproduction

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“…Competition binding experiments were performed with either europium-labeled H2 relaxin (26) or Eu 3ϩ -labeled INSL3 (27) in the absence or presence of increasing concentrations of unlabeled hormones. Nonspecific binding was determined with an excess of unlabeled peptides (500 nM H2 relaxin or INSL3).…”

Section: Methodsmentioning

confidence: 99%

The Minimal Active Structure of Human Relaxin-2

Hossain

Rosengren

Samuel

et al. 2011

Journal of Biological Chemistry

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H2 relaxin is a peptide hormone associated with a number of therapeutically relevant physiological effects, including regulation of collagen metabolism and multiple vascular control pathways. It is currently in phase III clinical trials for the treatment of acute heart failure due to its ability to induce vasodilation and influence renal function. It comprises 53 amino acids and is characterized by two separate polypeptide chains (A-B) that are cross-linked by three disulfide bonds. This size and complex structure represents a considerable challenge for the chemical synthesis of H2 relaxin, a major limiting factor for the exploration of modifications and derivatizations of this peptide, to optimize effect and drug-like characteristics. To address this issue, we describe the solid phase peptide synthesis and structural and functional evaluation of 24 analogues of H2 relaxin with truncations at the termini of its peptide chains. We show that it is possible to significantly truncate both the N and C termini of the B-chain while still retaining potent biological activity. This suggests that these regions are not critical for interactions with the H2 relaxin receptor, RXFP1. In contrast, truncations do reduce the activity of H2 relaxin for the related receptor RXFP2 by improving RXFP1 selectivity. In addition to new mechanistic insights into the function of H2 relaxin, this study identifies a critical active core with 38 amino acids. This minimized core shows similar antifibrotic activity as native H2 relaxin when tested in human BJ3 cells and thus represents an attractive receptor-selective lead for the development of novel relaxin therapeutics.Human relaxin-2 or H2 relaxin is a peptide hormone with multiple pleiotropic actions (1). Initially thought to be only a reproductive hormone involved in facilitating delivery of the young, more recent studies have demonstrated that H2 relaxin plays a key role in inflammatory and matrix remodeling processes and possesses potent vasodilatory, angiogenic, and other cardioprotective actions (2). The vasodilatory effects of H2 relaxin are thought to involve promotion of nitric oxide and the gelatinases, matrix metalloproteinase-2 and matrix metalloproteinase-9, in addition to antagonism of the vasoconstricting actions of endothelin-1 and angiotensin II (3). This causes systemic and renal vasodilation, increased arterial compliance, and other vascular changes. These findings have led to current clinical trial evaluation of relaxin as drug for the treatment of patients with acute heart failure (AHF) 6 (4, 5). Furthermore, the matrix remodeling actions of H2 relaxin have enhanced its reputation as a rapidly acting but safe antifibrotic agent, which has been further supported by its ability to successfully inhibit and/or reverse fibrosis in every preclinical model of experimental disease evaluated to date (6, 7).All of the above actions of H2 relaxin are thought to be mediated through its native receptor RXFP1 (originally named LGR7), which is a leucine-rich repeat containing G-prote...

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Solid-Phase Synthesis of Europium-Labeled Human INSL3 as a Novel Probe for the Study of Ligand−Receptor Interactions

Cited by 56 publications

References 38 publications

Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment

Prolonged RXFP1 and RXFP2 signaling can be explained by poor internalization and a lack of β-arrestin recruitment

Functional link between bone morphogenetic proteins and insulin-like peptide 3 signaling in modulating ovarian androgen production

The Minimal Active Structure of Human Relaxin-2

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