CORTICOTROPIN-RELEASING HORMONE-RECEPTOR TYPE-1 - GENERATION AND CHARACTERIZATION OF POLYCLONAL ANTIPEPTIDE ANTIBODIES AND THEIR LOCALIZATION IN PITUITARY-CELLS AND CORTICAL-NEURONS IN-VITRO

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stre ss. CRH acts by first binding to specific receptors on the plasma memb rane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we ha ve chosen three different peptide sequences within the CRH-R1 molecule which bear no similarity to other members of this receptor subfamily (or indeed any known protein) and which are likely to be exposed on th e surface of the native protein, for antibody production. Some of thes e fragments produced antipeptide antibodies of good titre which cross- reacted with the CRH-R1 receptor expressed in transiently transfected COS-7 cells and in tissue extracts from rat cerebellum, cortex, pituit ary gland and human myometrium, both in Western blots and in liquid-ph ase radioimmunoassay. We used immunofluorescence techniques to localiz e the CRH receptor in transiently transfected COS-7 cells, primary cul tures of rat anterior pituitary (AP) cells, the corticotroph-tumour ce lls AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasm a membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells thei r appearance is a fine punctate pattern, in AtT20 cells, they appear a s large patches which could account for receptor clusters. Within prim ary cortical neurons, their distribution does not appear to be polariz ed. Our results suggest that distribution of CRH-R1 receptors within t he different cell-types investigated depends not only on the amino aci d sequence but also on cellular factors.