Design and validation of recombinant protein standards for quantitative Western blot analysis of cannabinoid CB1 receptor density in cell membranes: an alternative to radioligand binding methods

被引：3

作者：

Saumell-Esnaola, Miquel ^{[1
,2
]}

Elejaga-Jimeno, Ainhoa ^{[3
]}

Echeazarra, Leyre ^{[4
,5
]}

Borrega-Roman, Leire ^{[1
,2
]}

Barrondo, Sergio ^{[1
,2
,6
]}

Lopez de Jesus, Maider ^{[1
,2
]}

Gonzalez-Burguera, Imanol ^{[2
,7
]}

Gomez-Caballero, Alberto ^{[3
]}

Goicolea, Maria Aranzazu ^{[3
]}

Salles, Joan ^{[1
,2
,6
]}

Garcia del Cano, Gontzal ^{[2
,7
]}

机构：

[1] Univ Basque Country UPV EHU, Fac Pharm, Dept Pharmacol, Vitoria 01006, Spain

[2] Bioaraba, Neurofarmacol Celular & Mol, Vitoria 01008, Spain

[3] Univ Basque Country UPV EHU, Fac Pharm, Dept Analyt Chem, Vitoria 01006, Spain

[4] Univ Basque Country UPWEHU, Fac Pharm, Dept Physiol, Vitoria 01006, Spain

[5] Bioaraba, Dispositivos Moviles El Control Enfermedades Cron, Vitoria 01008, Spain

[6] Ctr Invest Biomed Red Salud Mental CIBERSAM, Madrid 28029, Spain

[7] Univ Basque Country UPV EHU, Fac Pharm, Dept Neurosci, Vitoria 01006, Spain

来源：

MICROBIAL CELL FACTORIES | 2022年 / 21卷 / 01期

关键词：

GPCR expression analysis; Quantitative Western blot; Radioligand saturation binding; Cannabinoid CB1 receptor antibodies; Carboxy-terminal tail; Soluble recombinant protein standards; GST fusion proteins; PHOSPHOLIPASE-C-BETA; INVERSE AGONIST; SELECTIVE ANTAGONIST; TERNARY COMPLEX; PREFRONTAL CORTEX; TYPE-1; RECEPTOR; HUMAN BRAIN; RAT-BRAIN; LOCALIZATION; EXPRESSION;

D O I：

10.1186/s12934-022-01914-1

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

Background: Replacement of radioligand binding assays with antibody-antigen interaction-based approaches for quantitative analysis of G protein-coupled receptor (GPCR) levels requires the use of purified protein standards containing the antigen. GPCRs in general and cannabinoid CB1 receptor in particular show a progressive tendency to aggregate and precipitate in aqueous solution outside of their biological context due to the low solubility that the hydrophobic nature imprinted by their seven transmembrane domains. This renders full-length recombinant GPCRs useless for analytical purposes, a problem that can be overcome by engineering soluble recombinant fragments of the receptor containing the antigen. Results: Here we generated highly soluble and stable recombinant protein constructs GST-CB1(41)(4-)(472 )and GST-CB1(414-442) containing much of the human CB1 receptor C-terminal tail for use as standard and negative control, respectively, in quantitative Western blot analysis of CB1 receptor expression on crude synaptosomes of the adult rat brain cortex. To this end we used three different antibodies, all raised against a peptide comprising the C-terminal residues 443-473 of the mouse CB1 receptor that corresponds to residues 442-472 in the human homolog. Estimated values of CB1 receptor density obtained by quantitative Western blot were of the same order of magnitude but slightly higher than values obtained by the radioligand saturation binding assay. Conclusions: Collectively, here we provide a suitable Western blot-based design as a simple, cost-effective and radioactivity-free alternative for the quantitative analysis of CB1 receptor expression, and potentially of any GPCR, in a variety of biological samples. The discrepancies between the results obtained by quantitative Western blot and radioligand saturation binding techniques are discussed in the context of their particular theoretical bases and methodological constraints.

引用

页数：18

共 96 条

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