Analysis of the Cell Surface Expression of Cytokine Receptors Using the Surface Protein Biotinylation Method

Pavel, Mahmud Arif; Lam, Clarissa; Kashyap, Parul; Salehi-Najafabadi, Zahra; Singh, Gurpreet; Yu, Yong

doi:10.1007/978-1-4939-0928-5_16

Cited by 13 publications

(11 citation statements)

References 11 publications

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“…Cell surface biotinylation was performed as described by Pavel et al (2014). In short, COS-7 cells were transfected with pRK5 expression plasmids with FuGENE HD Transfection Reagent.…”

Section: Star Methodsmentioning

confidence: 99%

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation

Cho

Smallwood

Nathans

2017

Neuron

164

155

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Summary Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck combined with loss of Norrin impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck; and a soluble Reck(CC1–5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell-surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB.

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“…Cell surface biotinylation was performed as described by Pavel et al (2014). In short, COS-7 cells were transfected with pRK5 expression plasmids with FuGENE HD Transfection Reagent.…”

Section: Star Methodsmentioning

confidence: 99%

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation

Cho

Smallwood

Nathans

2017

Neuron

164

155

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“…Selection was performed by competitive elution with an excess of Enbrel on biotinylated TNF, captured by immobilized streptavidin (27). The principle of competitive elution is based on saturating all receptor binding sites on the cytokine, thereby preventing rebinding of dissociated phage antibodies, and thus enrichment for antagonistic VHH.…”

Section: Resultsmentioning

confidence: 99%

Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

et al. 2017

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The activity of tumor necrosis factor (TNF), a cytokine involved in inflammatory pathologies, can be inhibited by antibodies or trap molecules. Herein, llama-derived variable heavy-chain domains of heavy-chain antibody (VHH, also called Nanobodies™) were generated for the engineering of bivalent constructs, which antagonize the binding of TNF to its receptors with picomolar potencies. Three monomeric VHHs (VHH#1, VHH#2, and VHH#3) were characterized in detail and found to bind TNF with sub-nanomolar affinities. The crystal structures of the TNF–VHH complexes demonstrate that VHH#1 and VHH#2 share the same epitope, at the center of the interaction area of TNF with its TNFRs, while VHH#3 binds to a different, but partially overlapping epitope. These structures rationalize our results obtained with bivalent constructs in which two VHHs were coupled via linkers of different lengths. Contrary to conventional antibodies, these bivalent Nanobody™ constructs can bind to a single trimeric TNF, thus binding with avidity and blocking two of the three receptor binding sites in the cytokine. The different mode of binding to antigen and the engineering into bivalent constructs supports the design of highly potent VHH-based therapeutic entities.

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“…Proteins on Xenopus oocyte plasma membrane were detected with a Pierce Cell Surface Protein Isolation Kit by following a slightly modified protocol as described previously (83). In brief, 3 or 4 d after cRNA injection, oocytes (20 oocytes per reaction) were washed with ice-cold calcium-free oocyte Ringer's solution (OR2) [82.4 mM NaCl, 2.5 mM KCl, 1 mM MgCl 2 , 10 mM Hepes (pH 7.6)].…”

Section: Methodsmentioning

confidence: 99%

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant

Pavel

et al. 2016

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

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Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/ polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca 2+, inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing singlepoint ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.autosomal dominant polycystic kidney disease | TRP channels | TRPP2 | polycystin | gain of function

show abstract

Analysis of the Cell Surface Expression of Cytokine Receptors Using the Surface Protein Biotinylation Method

Cited by 13 publications

References 11 publications

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation

Bivalent Llama Single-Domain Antibody Fragments against Tumor Necrosis Factor Have Picomolar Potencies due to Intramolecular Interactions

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant

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