Site-selective labeling and electron paramagnetic resonance studies of human cannabinoid receptor CB2.
Biochim Biophys Acta Biomembr
; 1863(8): 183621, 2021 08 01.
Article
em En
| MEDLINE
| ID: mdl-33865808
ABSTRACT
Integral membrane G protein-coupled receptors (GPCR) regulate multiple physiological processes by transmitting signals from extracellular milieu to intracellular proteins and are major targets of pharmaceutical drug development. Since GPCR are inherently flexible proteins, their conformational dynamics can be studied by spectroscopic techniques such as electron paramagnetic resonance (EPR) which requires selective chemical labeling of the protein. Here, we developed protocols for selective chemical labeling of the recombinant human cannabinoid receptor CB2 by judiciously replacing naturally occurring reactive cysteine residues and introducing a new single cysteine residue in selected positions. The majority of the 47 newly generated single cysteine constructs expressed well in E. coli cells, and more than half of them retained high functional activity. The reactivity of newly introduced cysteine residues was assessed by incorporating nitroxide spin label and EPR measurement. The conformational transition of the receptor between the inactive and activated form were studied by EPR of selectively labeled constructs in the presence of either a full agonist CP-55,940 or an inverse agonist SR-144,528. We observed evidence for higher mobility of labels in the center of internal loop 3 and a structural change between agonist vs. inverse agonist-bound CB2 in the extracellular tip of transmembrane helix 6. Our results demonstrate the utility of EPR for studies of conformational dynamics of CB2.
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Base de dados:
MEDLINE
Assunto principal:
Conformação Proteica
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Espectroscopia de Ressonância de Spin Eletrônica
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Receptores de Canabinoides
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Receptor CB2 de Canabinoide
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article