Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Shabanpoor, Fazel; Bathgate, Ross A. D.; Belgi, Alessia; Chan, Linda J.; Nair, Vinojini B.; Wade, John D.; Hossain, Mohammed Akhter

doi:10.1016/j.bbrc.2012.02.141

Cited by 40 publications

(26 citation statements)

References 21 publications

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“…Comparison of ligand affinity for mutant receptors with wild-type RXFP2 was assessed using Europium (Eu 3+ )-labelled INSL3 (Eu-INSL3) 53 or H2 relaxin (Eu-H2) 54 at increasing concentrations in the presence or absence of 1 µM unlabeled ligand. Following a 1 h incubation, media was removed and 100 µl Delfia Enhancement solution (PerkinElmer) was added to each well.…”

Section: Methodsmentioning

confidence: 99%

Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Bruell

Sethi

Smith

et al. 2017

Sci Rep

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Relaxin family peptide receptor 2 (RXFP2) is a GPCR known for its role in reproductive function. It is structurally related to the human relaxin receptor RXFP1 and can be activated by human gene-2 (H2) relaxin as well as its cognate ligand insulin-like peptide 3 (INSL3). Both receptors possess an N-terminal low-density lipoprotein type a (LDLa) module that is necessary for activation and is joined to a leucine-rich repeat domain by a linker. This linker has been shown to be important for H2 relaxin binding and activation of RXFP1 and herein we investigate the role of the equivalent region of RXFP2. We demonstrate that the linker’s highly-conserved N-terminal region is essential for activation of RXFP2 in response to both ligands. In contrast, the linker is necessary for H2 relaxin, but not INSL3, binding. Our results highlight the distinct mechanism by which INSL3 activates RXFP2 whereby ligand binding mediates reorientation of the LDLa module by the linker region to activate the RXFP2 transmembrane domains in conjunction with the INSL3 A-chain. In contrast, relaxin activation of RXFP2 involves a more RXFP1-like mechanism involving binding to the LDLa-linker, reorientation of the LDLa module and activation of the transmembrane domains by the LDLa alone.

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Section: Methodsmentioning

confidence: 99%

Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Bruell

Sethi

Smith

et al. 2017

Sci Rep

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“…We performed competition binding experiments utilizing HEK-293T cells stably transfected with RXFP1 and europium-labeled human relaxin-2 according to Shabanpoor et al [36]. Briefly, cells were plated out in 96-well Isoplates with white walls and clear bottoms precoated with poly-L-lysine.…”

Section: Methodsmentioning

confidence: 99%

Relaxin-2 Does Not Ameliorate Nephropathy in an Experimental Model of Type-1 Diabetes

Dschietzig

Krause-Relle

Hennequin

et al. 2015

Kidney Blood Press Res

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Background/Aims: In diabetic nephropathy (DN), the current angiotensin-II-blocking pharmacotherapy is frequently failing. For diabetic cardiomyopathy (DC), there is no specific remedy available. Relaxin-2 (Rlx) - an anti-fibrotic, anti-inflammatory, and vasoprotecting peptide - is a candidate drug for both. Methods: Low-dose (32 µg/kg/day) and high-dose (320 µg/kg/day) Rlx were tested against vehicle (n = 20 each) and non-diabetic controls (n = 14) for 12 weeks in a model of type-1 diabetes induced in endothelial nitric oxide synthase knock-out (eNOS-KO) mice by intraperitoneal injection of streptozotocin. Results: Diabetic animals showed normal plasma creatinine, markedly increased albuminuria and urinary malonyldialdehyde, elevated relative kidney weight, glomerulosclerosis, and increased glomerular size, but no relevant interstitial fibrosis. Neither dose of Rlx affected these changes although the drug was active and targeted plasma levels were achieved. Of note, we found no activation of the renal TGF-β pathway in this model. In the hearts of diabetic animals, no fibrotic alterations indicative of DC could be determined which precluded testing of the initial hypothesis. Conclusions: We investigated a model showing early DN without overt tubulo-interstitial fibrosis and activation of the TGF-β-Smad-2/3 pathway. In this model, Rlx proved ineffective; however, the same may not apply to other models and types of diabetes.

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“…Eu-labeled H2 Relaxin Binding Assays-Saturation binding assays were performed on whole cells as described previously, using Europium labeled H2 relaxin (Eu-H2 relaxin) (25). Increasing concentrations of Eu-H2 relaxin (0.1-50 nM) were utilized for saturation binding, and nonspecific binding was determined in presence of 1 M of unlabeled H2 relaxin.…”

Section: Methodsmentioning

confidence: 99%

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

Diepenhorst

Petrie²,

Chen

et al. 2014

Journal of Biological Chemistry

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Background: Extracellular loops of the transmembrane domain of the relaxin receptor RXFP1 are predicted to interact with relaxin. Results: RXFP1 extracellular loops displayed on a scaffold protein enabled investigation of ligand interactions. Conclusion: RXFP1 activation involves interactions between the extracellular loops with relaxin and the receptor LDLa module. Significance: Understanding the molecular mechanisms of RXFP1 activation will aid drug design at this receptor.

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Site-specific conjugation of a lanthanide chelator and its effects on the chemical synthesis and receptor binding affinity of human relaxin-2 hormone

Cited by 40 publications

References 21 publications

Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Distinct activation modes of the Relaxin Family Peptide Receptor 2 in response to insulin-like peptide 3 and relaxin

Relaxin-2 Does Not Ameliorate Nephropathy in an Experimental Model of Type-1 Diabetes

Investigation of Interactions at the Extracellular Loops of the Relaxin Family Peptide Receptor 1 (RXFP1)

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