Solubilization and isolation of the human placenta receptor for epidermal growth factor-urogastrone.
We describe the solubilization and purification of both the photoaffinity-labeled and unlabeled human placenta receptor for epidermal growth factor-urogastrone (EGF-URO). The photolabeled receptor can be purified 300- to 500-fold from membrane extracts by combined immunoaffinity and lectin-agarose affinity chromatography. This isolation approaches theoretical purity. Upon gel filtration and wheat germ agglutinin-Sepharose chromatography, the photolabeled receptor and the EGF-URO binding activity co-migrate, as measured by a newly developed lectin-agarose immobilization assay of receptor binding. Sequential ion exchange, Cibacron blue-Sepharose, wheat germ agglutinin-Sepharose, and gel filtration chromatography yield a 110-fold purification of the EGF-URO binding activity; this purified fraction contains protein constituents that exhibit electrophoretic mobilities parallel to those of the photolabeled receptor constituents, that have apparent molecular weights of 180,000 and 160,000. This molecular weight range is consistent with a measured apparent Stokes radius for both the photolabeled and unlabeled receptor of 5.1 nm (Sephacryl S-200 gel filtration), although an apparent radius of 4.3 nm is estimated by gel filtration on Sepharose-6B. The apparent molecular weight of the photolabeled receptor is unaffected by 2-mercaptoethanol. This work provides a basis for further detailed studies of this growth factor receptor.
D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors; D005260:Female; D005768:Gastrointestinal Hormones / Q000378:metabolism*; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism*; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D012995:Solubility
D000345:Affinity Labels; D005768:Gastrointestinal Hormones; D010455:Peptides; D011956:Receptors, Cell Surface; D004815:Epidermal Growth Factor; D066246:ErbB Receptors