Functional characterization and purification of the secretin receptor expressed in baculovirus-infected insect cells.
Structural insights into Class II G protein-coupled receptors have been limited by the absence of a plentiful and highly enrichable source such as rhodopsin in the Class I family. With structural differences predicted to exist between these families, and with the key importance of an intact, disulfide-bonded amino-terminal domain for the Class II receptors, an overproduction and purification scheme is critically important. In this work, we have established and characterized a baculoviral expression and purification system for the secretin receptor. Hemagglutinin epitope-tagged wild-type rat secretin receptor construct was expressed using the recombinant baculovirus/Sf9 insect cell-based system, achieving a level of expression substantially higher than that previously achieved in Chinese hamster ovary (CHO-SecR) cells. Receptor expressed in Sf9 cells had similar affinity for secretin (Ki=1.4+/-0.2 nM) and similar potency to stimulate intracellular cAMP in response to this hormone (EC50=194+/-45 pM) as did wild-type receptor expressed in CHO cells. Receptors from Sf9 cells were also affinity labeled saturably and specifically by a photolabile secretin analogue. The receptors were purified to homogeneity by solubilization with sodium deoxycholate, selective ammonium sulfate precipitation, gel filtration and immunoaffinity purification. This expression system should facilitate the structural characterization of this receptor and its important amino-terminal domain.
Asmann
Yan W
YW
Cancer Center and Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic Scottsdale, Johnson Research Building, Scottsdale, AZ 85259, USA.
Dong
Maoqing
M
Miller
Laurence J
LJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000345:Affinity Labels; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D015870:Gene Expression; D066298:In Vitro Techniques; D007700:Kinetics; D051381:Rats; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012633:Secretin / Q000378:metabolism; D018411:Spodoptera
D000345:Affinity Labels; D043562:Receptors, G-Protein-Coupled; D011964:Receptors, Gastrointestinal Hormone; D011994:Recombinant Proteins; C029937:secretin receptor; D012633:Secretin; D000242:Cyclic AMP; D000262:Adenylyl Cyclases