High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

doi:10.1016/S0196-9781(99)00136-9

Peptides

Volume 20, Issue 11, November 1999, Pages 1311-1319

https://doi.org/10.1016/S0196-9781(99)00136-9 Get rights and content

Abstract

The structure-activity relationship (SAR) between the recently identified neuropeptide urocortin (Ucn) and corticotropin-releasing factor (CRF) receptor, type 1 (CRFR1), has been investigated. To this end, rat Ucn (rUcn), ovine CRF (oCRF) and chimeric peptides of rUcn and oCRF were synthesized and tested for their binding affinity and potency to stimulate cAMP production in human embryonic kidney (HEK) 293 cells stably transfected with cDNA encoding rat CRFR1 (rCRFR1). In binding studies with [¹²⁵I-Tyr⁰]oCRF or [³H-Leu⁹]rUcn as radioligand, it was observed that rUcn but not oCRF bound in a similar fashion as the CRF antagonist astressin with high affinity to rCRFR1 coupled to G protein or uncoupled from G protein by guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Consequently, rUcn was found to exert a significantly lower potency than oCRF to stimulate cAMP accumulation in transfected cells. CD spectroscopic investigations and reverse-phase HPLC (RPHPLC) retention behavior of the peptides suggested a more pronounced amphipatic α-helical character of rUcn when compared to oCRF and the chimeric peptides.

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 G_s 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, Nle^21,38, 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.

References (46)

C. Bergwitz et al.
Full activation of chimeric receptors by hybrids between parathyroid hormone and calcitonin
J Biol Chem
(1996)
C.P. Chang et al.
Identification of a seven transmembrane helix receptor for corticotropin-releasing factor and sauvagine in mammalian brain
Neuron
(1993)
P. Gourlet et al.
The C terminus ends of secretin and VIP interact with the N-terminal domains of their receptors
Peptides
(1996)
D. Hoyer et al.
Partial agonists, full agonists, antagonistsdilemmas of definition
Trends Pharmacol Sci
(1993)
T. Kenakin
Differences between natural and recombinant G protein-coupled receptor systems with varying receptor/G protein stoichiometry
Trends Pharmacol Sci
(1997)
Y. Oki et al.
Distribution and concentration of urocortin, and effect of adrenalectomy on its content in rat hypothalamus
Life Sci
(1998)
S.W. Provencher
A constrained regularization method for inverting data represented by linear algebraic or integral equations
Comput Phys Commun
(1982)
O. Valdenaire et al.
A new functional isoform of the human CRF2 receptor for corticotropin-releasing factor
Biochim Biophys Acta
(1997)
N. Vita et al.
Primary structure and functional expression of mouse and human brain corticotropin releasing factor receptors
FEBS Lett
(1993)
B. Wulff et al.
The C terminal part of VIP is important for receptor binding and activation, as evidenced by chimeric constructs of VIP/secretin
FEBS Lett
(1997)

D.P. Behan et al.

Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding proteinimplications for the treatment of CNS disorders

Mol Psychiatry

(1996)

M. Beyermann et al.

A single-point slight alteration set as a tool for structure-activity relationship studies of ovine corticotropin releasing factor

J Med Chem

(1996)

R. Chen et al.

Expression cloning of a human corticotropin-releasing-factor receptor

Proc Natl Acad Sci USA

(1993)

P.Y. Chou et al.

Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins

Biochemistry

(1974)

F.M. Dautzenberg et al.

Identification of two corticotropin-releasing factor receptors from xenopus laevis with high ligand selectivityunusual pharmacology of the type 1 receptor

J Neurochem

(1997)

E.B. De Souza et al.

Corticotropin-releasing factor receptors in human pituitary glandautoradiographic localization

Neuroendocrinol

(1985)

C.J. Donaldson et al.

Cloning and characterization of human urocortin

Endocrinology

(1996)

B. Fichtl et al.

Wirkungen von Pharmaka auf den Organismus

J. Gulyas et al.

Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor

Proc Natl Acad Sci USA

(1995)

J.F. Hernandez et al.

Synthesis and relative potencies of new constrained CRF antagonists

J Med Chem

(1993)

K. Iino et al.

Urocortin expression in human pituitary gland and pituitary adenoma

J Clin Endocrin Metab

(1997)

T. Kishimoto et al.

A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle

Proc Natl Acad Sci USA

(1995)

W.D. Kornreich et al.

Alanine series of ovine corticotropin releasing factor (oCRF)a structure-activity relationship study

J Med Chem

(1992)

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Corticotropin-releasing factor (CRF) and the CRF-like peptide urocortin I (UcnI) exert their activity through two different CRF receptors, CRF₁ and CRF₂. Recently, UcnII and UcnIII have been discovered as potential endogenous agonists selective for CRF₂ known to be involved in brain functions such as learning and anxiety, as well as in cardiovascular functions. A structure–affinity relationship study using chimeric peptides was designed to characterize mouse UcnII (mUcnII) and mUcnIII further and to investigate the structural basis of their receptor subtype selectivity. In the framework of this study, mUcnII (IC₅₀=4.4 nM) but not mUcnIII was identified as high-affinity ligand for the rat CRF binding protein. Such affinity had previously not been observed for the human version of this protein. On the basis of secondary structure predictions, it was hypothesized that the amino acid motifs Pro-Ile-Gly of mUcnII and Pro-Thr-Asn of mUcnIII decrease α-helicity and thereby impair binding to CRF₁. In support of this hypothesis, binding affinity to CRF₁ of the chimeric peptides [Pro¹¹Ile¹²Gly¹³]h/rCRF, [Pro¹¹Thr¹²Asn¹³]h/rCRF, and the corresponding rUcnI analogs was found to be decreased by three orders of magnitude, whereas binding affinity to CRF₂ was much less affected. The dramatic decrease in binding affinity to CRF₁ correlated with a decrease in α-helicity as indicated by the data of circular dichroism spectroscopy.
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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.

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Regular papersHigh-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

Abstract

Introduction

Section snippets

Synthesis and analysis of peptides

Physicochemical characterization of peptides

Displacement of [125I-Tyr0]oCRF from recombinant rCRFR1 by CRF analogs in the absence and presence of GTPγS

Discussion

Acknowledgements

J Biol Chem

Neuron

Peptides

Trends Pharmacol Sci

Trends Pharmacol Sci

Life Sci

Comput Phys Commun

Biochim Biophys Acta

FEBS Lett

FEBS Lett

Neurobiology of corticotropin releasing factor (CRF) receptors and CRF-binding proteinimplications for the treatment of CNS disorders

Mol Psychiatry

A single-point slight alteration set as a tool for structure-activity relationship studies of ovine corticotropin releasing factor

J Med Chem

Expression cloning of a human corticotropin-releasing-factor receptor

Proc Natl Acad Sci USA

Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins

Biochemistry

Identification of two corticotropin-releasing factor receptors from xenopus laevis with high ligand selectivityunusual pharmacology of the type 1 receptor

J Neurochem

Corticotropin-releasing factor receptors in human pituitary glandautoradiographic localization

Neuroendocrinol

Cloning and characterization of human urocortin

Endocrinology

Wirkungen von Pharmaka auf den Organismus

Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor

Proc Natl Acad Sci USA

Synthesis and relative potencies of new constrained CRF antagonists

J Med Chem

Urocortin expression in human pituitary gland and pituitary adenoma

J Clin Endocrin Metab

A sauvagine/corticotropin-releasing factor receptor expressed in heart and skeletal muscle

Proc Natl Acad Sci USA

Alanine series of ovine corticotropin releasing factor (oCRF)a structure-activity relationship study

J Med Chem

Regular papers
High-affinity binding of urocortin and astressin but not CRF to G protein-uncoupled CRFR1

Displacement of [¹²⁵I-Tyr⁰]oCRF from recombinant rCRFR1 by CRF analogs in the absence and presence of GTPγS