Research reportStructure–function relationship of different domains of the rat corticotropin-releasing factor receptor
Introduction
Corticotropin-releasing factor (CRF) is a 41-amino acid peptide [36]which plays an important neuroendocrine role in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis [41]and is involved in the coordination of various responses to stress [44]. It acts via the secretion of hypophyseal adrenocorticotropic hormone (ACTH) which stimulates the release of adrenal glucocorticoids. In the brain, CRF induces different autonomic, electrophysiological and behavioral effects, indicating that this peptide serves as a neuromodulator 8, 30. Recent findings are also suggesting that CRF is involved in the regulation of the immune response to stress 6, 20.
CRF exhibits its effects through membrane-bound receptors. The CRF receptors (CRFRs) described to date possess seven putative transmembrane domains (TM), are coupled to guanine nucleotide stimulatory protein (GS) and stimulate adenylate cyclase. Two classes of mammalian CRFRs have been described: CRFR1 and CRFR2. CRFR1 has been cloned from human 5, 43, rat 4, 31, mouse [43]and chicken [46]origin. The receptor protein consists of 415–420 amino acids with five and six putative N-linked glycosylation sites in the N-terminal extracellular domain of the human and rodent protein, respectively. When the primary structures of the receptor molecules from different species were compared, a degree of identity of 98% was determined. Two splice variants, α and β, of CRFR2 are known. CRFR2α [25]comprises 411 amino acids and shares approximately 71% identity with CRFR1. The 431-amino acid sequence of CRFR2β differs from CRFR2α in the first 54 amino acids 21, 24, 25, 31, 37. Both CRFR2α and CRFR2β contain five putative N-glycosylation sites.
Several studies describe the molecular diversity and pharmacologic responses of CRFR and the localization of CRFR subtypes. In addition to autoradiographic binding studies [7], in situ hybridizations have been performed. mRNA molecules of CRFR1 and CRFR2 were found to be distributed heterogeneously in the brain [24]so that distinctive functional roles of the receptor proteins seem likely.
On the protein chemical level, most studies have been performed utilizing covalent labeling techniques. Molecular weights of approximately 70 000 have been reported for CRFR in pituitary 13, 15and spleen [44], whereas a 53 kDa receptor was detected in brain tissue 13, 15. The different molecular weights of CRFR point to a different regulation of the receptor modification in various tissues. Deglycosylation of CRFR proteins from both brain and pituitary yielded 40–45 kDa proteins [15]. This size was in agreement with the molecular weight predicted on the basis of cDNA data after removal of the signal sequence and without posttranslational modifications [5].
With regard to structure–function relationships, CRFRs are barely characterized to date. The involvement of extra- and intracellular domains of CRFR in ligand binding and G-protein coupling has not yet been established. The objective of the work presented here was to investigate the significance of the extra- and intracellular and transmembrane domains of CRFR1 for the activity and functionality of the receptor protein. The strategy of this investigation was to generate cDNA constructs coding for truncated CRFR1 molecules of different length. Purification and identification of the receptor analogs were facilitated by prolongation with nucleotides coding for six histidine residues used for affinity chromatography and immunodetection. We also present two polyclonal CRFR1 antibodies that enabled the analysis of the subcellular localization of CRFR1 in transfected mammalian cells.
Section snippets
Antigen production and purification
Two antigens were produced for the immunization of rabbits, one corresponding to the N-terminus of rCRFR1 (rCRFR1-I=rCRFR1-NT), the other to the C-terminus of rCRFR1 (rCRFR1-CT). The cDNA fragment coding for rCRFR1-NT was amplified by PCR and cloned into the procaryotic expression vector pQE30 (Qiagen, Hilden, Germany) utilizing the restriction enzymes BamHI and KpnI. The protein sequence of rCRFR1-NT was as follows: MRGSHHHHHHG
Antibody specificity
The DNA coding for the N-terminal part of rCRFR1 (rCRFR1-NT) was overexpressed in E. coli M15 cells. The protein production was induced by 1 mM IPTG (Fig. 1A). rCRFR1-NT, a 12.25 kDa protein, was purified to homogeneity with nickel-affinity chromatography (Fig. 1A). The purified protein was identified by its N-terminal sequence determined by Edman degradation and reversed-phase HPLC linked to electrospray mass spectrometry (LC-MS). On the basis of HPLC, no evidence for heterogeneity was
Specificity of the polyclonal antibodies anti-rCRFR1-NT and anti-rCRFR1-CT
In this study, we present two antibodies which were generated against the N- and C-terminal regions of rCRFR1. As demonstrated above, both antibodies interact specifically with rat and human CRFR1. The recognition of hCRFR1 by an antibody directed against the corresponding rat protein was not surprising in view of the high homology between both receptor molecules which differed by only 12 amino acids [32]. However, the antibodies could discriminate between CRFR1 and CRFR2β as demonstrated by
Acknowledgements
We gratefully acknowledge the excellent technical contributions of Gudrun Fricke-Bode and Andrea Flaccus. We also wish to thank Thomas Liepold for protein sequence analysis and for his participation in performing the cAMP assays. Further thanks go to Claudia Weber and Christian Ebeling who supported the work as students in the laboratory. We also thank Dr. Ulrich Teichert and Ulrike Schulz for oligonucleotide synthesis and Dr. Andreas Rühmann and Bernd Hesse for peptide synthesis. Dr. Klaus
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2003, Progress in Medicinal ChemistryCitation Excerpt :The role played by the NT domain in peptide ligand binding has been explored by using the isolated NT in binding and structural measurements. rCRF-NT devoid of the signal peptide (1–23) was expressed in Escherichia coli and bound oCRF specifically (over VIP) but with low affinity (IC50=6.8 μM) [38]. Progressive elongation of the NT region towards the C-terminus afforded a receptor which bound oCRF both with high affinity (IC50=61 nM) and the ability to stimulate cAMP accumulation only when the seventh helical domain was included, suggesting that while IC-3 is important for signalling, the NT domain can form productive-binding interactions with CRF by itself.