Expression and purification of Gpr33, a candidate membrane receptor for cortisol

Membrane receptors for the sex steroids progesterone, estrogen, and androgens have been identified, and membrane receptors for the adrenocortical steroids glucocorticoids and mineralocorticoids have also been suggested to exist but have not yet been identified. Because of their diverse physiological actions, corticoids have been artificially synthesized and widely used as steroid drugs, and membrane receptors for corticoids are expected to become new drug targets. Therefore, there is a need to identify membrane glucocorticoid receptors (mGRs). To screen candidate mGRs, we identified two common amino acid sequences between membrane progesterone receptor (mPR) and membrane estrogen receptor (mER). We searched the protein database PROSITE for these common sequences. This led us to select GPR33 as a candidate mGR. To determine whether GPR33 can bind to corticoids, recombinant mouse Gpr33 (MGpr33) was expressed in Pichia yeast. Recombinant MGpr33, which was present as an oligomer in the membrane fraction, was found to bind to cortisol (hydrocortisone), a type of glucocorticoid. These results verified that GPR33 is a candidate membrane corticoid receptor. Denaturation by urea resulted in the generation of MGpr33 monomers and reduced the cortisol-binding activity of MGpr33, suggesting that MGpr33 may function as an oligomer. MGpr33 oligomers purified by two-step column chromatography using a Ni-NTA column and a Sephacryl S-300 gel filtration column retained the ability to bind cortisol. The purified fraction bound to cortisol with a Kd = 11.9 nM and Bmax = 1.19 nM. Furthermore, MGpr33 specifically bound to cortisol but not to estradiol, progesterone or testosterone. MGpr33 was found to be a candidate mGR that may function as an oligomer.