Isolation of the human placenta receptor for epidermal growth factor-urogastrone

Abstract

EPIDERMAL growth factor-urogastrone (EGF-URO), a singlechain polypeptide of molecular weight ∼6,000, found both in the mouse^1–3 and in man^4–7, is both a potent stimulant of cell proliferation and an inhibitor of gastric acid secretion. Specific membrane receptors for EGF-URO can be detected in various tissues including human placenta^8–11. Although the mouse and human polypeptide sequences differ at 16 positions, they can share the same receptor site in human tissues^3,12. There has been considerable recent interest in EGF-URO and its receptor, not only because of the potent mitogenic and acid-inhibitory actions of the polypeptide, but also because of the association of viral¹³, chemical¹⁴ and spontaneous¹⁵ cell transformation with a specific reduction in cell receptors for EGF-URO. It is also believed that EGF-URO and its receptor may be involved in the development, maintenance, and differentiation of tissues as diverse as the lens¹⁶, the palate¹⁷ and the keratin layer of skin¹⁸. In experiments aimed at the isolation of the membrane receptor for EGF-URO, we have observed that, unlike the receptor for insulin¹⁹, but like the muscarinic receptor for acetylcholine²⁰, the EGF-URO receptor loses its ligand recognition property following solubilisation with detergents. We therefore developed an affinity-labelling technique using glutaraldehyde, that can covalently crosslink the receptor with ¹²⁵I-labelled EGF-URO before solubilisation²¹. Independently, Das and coworkers have developed a photoaffinity-labelling technique that can also be used to radiolabel the EGF-URO receptor before solubilisation²². Here we report the success of both our previously described glutaraldehyde crosslinking technique²¹ (using ¹²⁵I-EGF-URO for receptor labelling) and of a newly developed photoaffinity-labelling technique (using a photolabile ¹²⁵I-EGF-URO crosslinking reagent) to identify the EGF-URO receptor in human placenta membranes and to effect the isolation of the affinity-labelled receptor by affinity chromatography using lectin and murine EGF-URO antibody–Agarose columns.