Regular papersHigh-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1
Introduction
Corticotropin-releasing factor (CRF), believed to synchronize the endocrine, autonomic, immunologic and behavioral responses to stress, was characterized as a 41-residue polypeptide on the basis of its ability to stimulate the secretion of adrenocorticotropic hormone (ACTH) from the anterior pituitary [41].
CRF exhibits its activity through Gs protein-coupled receptors. CRF receptor, type 1 (CRFR1) mainly found in pituitary and brain was cloned from human, mouse, rat, chicken, and frog [4], [5], [7], [28], [43], [46]. cDNAs coding for two splice variants of CRF receptor, type 2, CRFR2α and CRFR2β, were cloned from brain, heart, and skeletal muscle [17], [21], [29], [37]. In rodents, CRFR2α has been exclusively found in the CNS, whereas CRFR2β is predominantly distributed in the periphery. In humans, both receptor subtypes have been found in the CNS [40]. Recently, it has been proposed that urocortin (Ucn), a natural CRF analog, is the endogenous ligand to CRFR2 [42].
After the discovery of Ucn and its co-localization with CRFR2 in distinct areas of the brain [42], Ucn mRNA has also been found in rat brain, pituitary [44] and human placenta [31], where CRFR1 is the predominant receptor [1]. Additionally, Ucn immunoreactivity was detected to a high degree in the pituitary gland from human [15] and rat [25] origin and in human placenta [31]. However, results obtained from different experiments in intact [39] and adrenalectomized rats [25] suggest that Ucn may not be a major endogenous hypothalamic hypophysiotropic regulator of pituitary ACTH secretion.
Binding studies using [Nle21,125I-Tyr32] ovine CRF ([Nle21, 125I-Tyr32]oCRF), [125I-Tyr0] rat Ucn ([125I-Tyr0]rUcn), and membranes of CHO cells stably transfected with cDNA coding for human CRFR1, rat CRFR2α or mouse CRFR2β revealed a distinct selectivity of human/rat CRF (h/rCRF) to be bound with high affinity to human CRFR1 [9]. In agreement with these data, h/rCRF was found to be less potent to stimulate cAMP production in cells expressing rat CRFR2α or mouse CRFR2β [9]. Interestingly, rUcn exhibited an equally high binding affinity to human CRFR1, rat CRFR2α or mouse CRFR2β and was shown to be equipotent to stimulate cAMP production in stably transfected cells expressing these receptors [9].
In a site-directed-mutagenesis approach with human CRFR1, it was found that the potency of Ucn to stimulate cAMP production in transfected cells—unlike the potency of CRF and sauvagine (Svg)—was not affected by a distinct replacement of amino acids in the second and third extracellular loop of the receptor with the corresponding amino acids of human CRFR2α. These data indicate a different binding mechanism of Ucn to human CRFR1 when compared to CRF or Svg [20].
Svg, white-suckerfish urotensin (sfUro), carp urotensin (cUro), h/rCRF, and oCRF reveals 35–63% amino acid identity. The consensus sequence of Svg, sfUro, cUro, h/rCRF, oCRF, and rUcn shows high similarity at the N-terminus (60%), but little at the C terminus (15%) of the peptides.
We used rUcn, oCRF, and two chimeric constructs of rUcn and oCRF, named rUcn(1–20)oCRF-(22–41) and oCRF-(1–20)rUcn-(20–40) (Fig. 1 ), to examine the ligand-receptor interaction of rUcn with rCRFR1. Binding of the peptides was tested in the absence or presence of guanosine 5′-O-(3-thiotriphosphate) (GTPγS) to distinguish between G protein-coupled and uncoupled receptors [22]. Agonist binding to G protein-coupled and uncoupled rCRFR1 was compared to the binding of astressin, {cyclo(30–33)[d-Phe12, Nle21,38, Glu30, Lys33]h/rCRF-(12–41)}, a potent CRF antagonist to CRFR1 [12] In addition, G protein-mediated adenylate cyclase activity after loading the receptor with ligand was measured in HEK 293 cells stably transfected with cDNA coding for rCRFR1 (HEK-rCRFR1 cells). Furthermore, reverse-phase HPLC (RPHPLC) retention times and CD spectroscopic data of the CRF peptides were compared with their binding and coupling efficiency.
Section snippets
Synthesis and analysis of peptides
The CRF like peptides were synthesized, purified, and analyzed as described [36].
Physicochemical characterization of peptides
For further characterization of the physicochemical properties of the pure peptides, all analogs were subjected to analytical RPHPLC on a Vydac C18 silica gel column (0.46 × 25 cm, 5-μM particle size, 30-nm pore size) with solvents A (0.1% TFA in water) and B (80% MeCN in 0.1% TFA in water) at a flow rate of 1 ml/min. The samples were eluted with 5% B for 5 min and then with a linear gradient of 5–95% B in 30 min.
Displacement of [125I-Tyr0]oCRF from recombinant rCRFR1 by CRF analogs in the absence and presence of GTPγS
The specific binding of [125I-Tyr0]oCRF and [3H-Leu9]rUcn to membranes of HEK-rCRFR1 cells was found to be 93% and 78%, and was decreased by 50% and 16%, respectively, when binding experiments were performed in the presence of GTPγS Fig. 2, Fig. 3, Fig. 4. GTPγS treatment reduced the specific binding of [3H-Leu9]rUcn to rCRFR1 to a lesser extent than the specific binding of [125I-Tyr0]oCRF to the same receptor Fig. 2, Fig. 3, Fig. 4.
In binding assays with [125I-Tyr0]oCRF and membranes of
Discussion
The fact that Ucn proposed as the natural high-affinity ligand of CRFR2 [42] was co-localized with CRFR1 in distinct areas of the brain and the periphery prompted us to a careful analysis of the ligand-receptor interaction between Ucn and CRFR1. Because oCRF exhibits low-affinity binding to CRFR2 and—unlike h/rCRF—does not bind to the CRF binding protein [1], oCRF and its 125I-iodinated analogs were used in binding and autoradiographic studies to label CRFR1 in membrane and tissue preparations
Acknowledgements
Supported by the Max Planck Society. Thomas Liepold is acknowledged for the performance of the amino acid analysis. Jürgen Schünemann is acknowledged for his support recording the circular dichroism spectra. We thank Dr Bodo Zimmermann for the performance of the mass spectrometric experiments. We are grateful to Dr C. Stevens and Dr G. Sharma for providing the HEK 293 cells.
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2004, Journal of Biological ChemistryCitation Excerpt :In other words, all peptides are likely to use or induce almost the same active receptor states and should not differ in their abilities to stimulate separate stimulus-response pathways via different G protein-coupled messenger systems. This conclusion is not in line with results showing that urocortin stimulated cAMP accumulation in HEK-CRFR1 cells with lower potency than oCRF (25) and that urocortin and CRF regulated differently the GRK3 activity in human retinoblastoma Y79 cells (26) and the mitogen-activated protein kinase signal transduction pathway in human pregnant myometrium and transfected cells (14). The reasons for these different results on agonist-specific trafficking of CRFR1 signaling remain unclear.
A soluble form of the first extracellular domain of mouse type 2β corticotropin-releasing factor receptor reveals differential ligand specificity
2003, Journal of Biological ChemistryCitation Excerpt :Another explanation for these observations is that sauvagine and urocortin III require correct coupling of the receptor to a G-protein, whereas urocortin I and urocortin II are able to bind with high affinity to receptors in absence of G-protein coupling. These data are consistent with the lack of effect of GTP on binding of urocortin I (21, 37) and with the suggestion that urocortin I may possess intrinsic antagonistic properties (38). The data in Table I show that the apparent affinities of urocortin III and of sauvagine for wild-type mCRFR2β depend on the nature of the labeled analog that is used in the radioreceptor assay.
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- 1
Present address: CME, Laboratory of Glycobiology and Developmental Genetics, Capus Gasthuisberg, K.U. Leuven Herestraat 49, B-3000 Leuven, Belgium.
- 2
Present address: Janssen Research Foundation, Collaboration and Technology Transfer, Turnhoutseweg 30, B-2340 Beerse, Belgium.