Dataset Information

Non-full-length Water-Soluble CXCR4^QTY and CCR5^QTY Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors.

ABSTRACT: It was posited that functionalities of GPCRs require full-length sequences that are negated by residue deletions. Here we report that significantly truncated nfCCR5^QTY and nfCXCR4^QTY still bind native ligands. Receptor-ligand interactions were discovered from yeast 2-hybrid screening and confirmed by mating selection. Two nfCCR5^QTY (SZ218a, SZ190b) and two nfCXCR4^QTY (SZ158a, SZ146a) were expressed in E. coli. Synthesized receptors exhibited ?-helical structures and bound respective ligands with reduced affinities. SZ190b and SZ158a were reconverted into non-QTY forms and expressed in HEK293T cells. Reconverted receptors localized on cell membranes and functioned as negative regulators for ligand-induced signaling when co-expressed with full-length receptors. CCR5-SZ190b individually can perform signaling at a reduced level with higher ligand concentration. Our findings provide insight into essential structural components for CCR5 and CXCR4 functionality, while raising the possibility that non-full-length receptors may be resulted from alternative splicing and that pseudo-genes in genomes may be present and functional in living organisms.

SUBMITTER: Qing R

PROVIDER: S-EPMC7756140 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Non-full-length Water-Soluble CXCR4QTY and CCR5QTY Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors.

Qing Rui R Tao Fei F Chatterjee Pranam P Yang Gaojie G Han Qiuyi Q Chung Haeyoon H Ni Jun J Suter Bernhard P BP Kubicek Jan J Maertens Barbara B Schubert Thomas T Blackburn Camron C Zhang Shuguang S

iScience 20201028 12

It was posited that functionalities of GPCRs require full-length sequences that are negated by residue deletions. Here we report that significantly truncated nfCCR5QTY and nfCXCR4QTY still bind native ligands. Receptor-ligand interactions were discovered from yeast 2-hybrid screening and confirmed by mating selection. Two nfCCR5QTY (SZ218a, SZ190b) and two nfCXCR4QTY (SZ158a, SZ146a) were expressed in E. coli. Synthesized receptors exhibited α-h ...[more]

PMID: 33376963

Similar Datasets

Project description:Chemokine receptors belong to a class of integral membrane G-protein coupled receptors (GPCRs) and are responsible for transmitting signals from the extracellular environment. However, the structural changes in the receptor, connecting ligand binding to G-protein activation, remain elusive for most GPCRs due to the difficulty to produce them for structural and functional studies. We here report high-level production in E.coli of 4 human GPCRs, namely chemokine receptors (hCRs) CCR5, CCR3, CXCR4 and CX3CR1 that are directly involved in HIV-1 infection, asthma and cancer metastasis. The synthetic genes of CCR5, CCR3, CXCR4 and CX3CR1 were synthesized using a two-step assembly/amplification PCR method and inserted into two different kinds of expression systems. After systematic screening of growth conditions and host strains, TB medium was selected for expression of pEXP-hCRs. The low copy number pBAD-DEST49 plasmid, with a moderately strong promoter tightly regulated by L-arabinose, proved helpful for reducing toxicity of expressed membrane proteins. The synthetic Trx-hCR fusion genes in the pBAD-DEST49 vector were expressed at high levels in the Top10 strain. After a systematic screen of 96 detergents, the zwitterionic detergents of the Fos-choline series (FC9-FC16) emerged as the most effective for isolation of the hCRs. The FC14 was selected both for solubilization from bacterial lysates and for stabilization of the Trx-hCRs during purification. Thus, the FC-14 solubilized Trx-hCRs could be purified using size exclusion chromatography as monomers and dimers with the correct apparent MW and their alpha-helical content determined by circular dichroism. The identity of two of the expressed hCRs (CCR3 and CCR5) was confirmed using immunoblots using specific monoclonal antibodies. After optimization of expression systems and detergent-mediated purification procedures, we achieved large-scale, high-level production of 4 human GPCR chemokine receptor in a two-step purification, yielding milligram quantities of CCR5, CCR3, CXCR4 and CX3CR1 for biochemical, biophysical and structural analysis.

Dataset Information

Non-full-length Water-Soluble CXCR4^QTY and CCR5^QTY Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors.

Publications

Non-full-length Water-Soluble CXCR4<sup>QTY</sup> and CCR5<sup>QTY</sup> Chemokine Receptors: Implication for Overlooked Truncated but Functional Membrane Receptors.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets