Corticotrophin‐Releasing Hormone Receptor Type 1: Generation and Characterization of Polyclonal Antipeptide Antibodies and their Localization in Pituitary Cells and Cortical Neurones in vitro

Abstract

Corticotrophin-releasing hormone (CRH) is a 41 amino acid neuropeptide which plays a major role in regulating the endocrine response to stress. CRH acts by first binding to specific receptors on the plasma membrane of target cells. A CRH receptor from a human corticotroph adenoma and rat brain has recently been cloned (CRH-R1). In this paper, we have 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 the surface of the native protein, for antibody production. Some of these 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, pituitary gland and human myometrium, both in Western blots and in liquid-phase radioimmunoassay. We used immunofluorescence techniques to localize the CRH receptor in transiently transfected COS-7 cells, primary cultures of rat anterior pituitary (AP) cells, the corticotroph-tumour cells AtT20 D16-16 and cortical neurons in primary culture. Our results indicate IR-CRH-R1 receptors have a punctate distribution on the plasma membrane of AP cells and AtT20 D16-16 cells. Whilst in AP cells their appearance is a fine punctate pattern, in AtT20 cells, they appear as large patches which could account for receptor clusters. Within primary cortical neurons, their distribution does not appear to be polarized. Our results suggest that distribution of CRH-R1 receptors within the different cell-types investigated depends not only on the amino acid sequence but also on cellular factors.

Corticotropin-Releasing Hormone Type I Receptor Messenger Ribonucleic Acid and Protein Levels in the Ovine Fetal Pituitary: Ontogeny and Effect of Chronic Cortisol Administration

“…Castro and colleagues reported a molecular mass for rat CRH R1 receptor in the pituitary ranging from 115-40 kDa (22). More recent studies have demonstrated sizes of 72 and 59 kDa in the mouse and rat (40).…”

“…In addition, CRH R1 receptor protein levels decreased after the precocious elevation of plasma cortisol levels in fetuses before term. In the aforementioned studies, CRH R1 receptor protein with molecular masses of 40, 45, 59, 72, 99, 110, and 115 kDa (22,40) have been explained as potentially due to differences in glycosylation and alternative splicing and as a function of the quality of the membrane preparation (22,40). It is possible that the different molecular masses observed in the present study may be related to the ovine species, as no other studies have reported the molecular size of the CRH R1 in the sheep.…”

“…1), was identified with enhanced chemiluminescence (ECL Plus, Amersham Pharmacia Biotech, Arlington Heights, IL), and relative intensity was normalized by the intensity of the internal control adult pituitary membrane preparation (PelFreez, Rogers, AR). The specificity of the primary antibody and validation of its use for detecting the presence of the CRH R1 receptor have been previously described (22).…”

Characterization of native corticotropin-releasing factor receptor type 1 (cRFR1) in the rat and mouse central nervous system

“…Previous reports describing molecular weights of CRFR1 from brain were controversial. By Western blot analysis using another polyclonal antibody against CRFR1 (Castro et al, 1996), 115-40 kDa forms of CRFR1 were found. On the basis of chemical cross-linking experiments, a 53 kDa form has 11.1 Ϯ 4.6 9.8 Ϯ 2.3 10.8 Ϯ 3.9 12.1 Ϯ 3.1 Statistically significant differences: *P Ͻ 0.05, **P Ͻ 0.01, ***P Ͻ 0.001 when compared to the corresponding value of the rat.…”

Dimerization Between Vasopressin V1b and Corticotropin Releasing Hormone Type 1 Receptors

“…The predicted molecular weights (MW) of both CRHR1 and V1b receptors are 40-45 kilo Dalton (kDa). However, CRHR1 Western Blots show large molecular weight bands of 118 and 200 kDa in sheep anterior pituitaries (Green et al, 2000) and 115 kDa in rat anterior pituitaries (Castro et al, 1996). In addition, solubilization of radiolabeled CRH receptors in rat pituitary shows bands of 100-130 kDa in addition to a major band of 75 kDa (Grigoriadis and De Souza, 1988;Flores et al, 1990).…”