Expression, purification and in vitro functional reconstitution of the chemokine receptor CCR1.
Chemokine receptors are a specific class of G-protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-beta-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a K(d) of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of approximately 0.1-0.2mg of pure functional receptor per 10(9) cells will permit biophysical studies of this medically important receptor.
Allen
Samantha J
SJ
Skaggs School of Pharmacy and Pharmaceutical Science, University of California-San Diego, La Jolla, CA 92093-0684, USA. samantha_allen@merck.com
Ribeiro
Sofia
S
Horuk
Richard
R
Handel
Tracy M
TM
D000595:Amino Acid Sequence; D002460:Cell Line; D018925:Chemokines / Q000378:metabolism; D019742:Chemokines, CC / Q000235:genetics / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006801:Humans; D008823:Micelles; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D054389:Receptors, CCR1 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*
C097720:CCL14 protein, human; D018925:Chemokines; D019742:Chemokines, CC; D003902:Detergents; D008823:Micelles; D054389:Receptors, CCR1