Differential responsiveness of CRF receptor subtypes to N-terminal truncation of peptidic ligands
Introduction
Corticotropin-releasing factor (CRF), a 41 amino acid peptide [31], stimulates the secretion of adrenocorticotropin [33] and acts as an early endocrine signal in the stress response (reviewed in [10], [18], [29]). It plays a modulating role in several brain functions and is linked to various neuropsychiatric conditions [1]. CRF and CRF-like peptides exert their effects by binding to G protein-coupled receptors, resulting in increased cAMP levels [5]. The cDNA coding for two CRF receptor subtypes, CRF1 and CRF2 receptors, has been cloned and characterized from various species (reviewed in [8], [30], [34]). Three different splice variants of CRF2 receptor have been described, CRF2α, CRF2β and CRF2γ [14], [16], [19]. CRF1 and CRF2 receptor display approximately 70% sequence homology. However, they differ from one another in their ligand specificity. Cells producing mammalian CRF1 receptor [5], [21], [36] can be activated by CRF, the frog skin peptide sauvagine (Svg) [11] and the CRF-related neuropeptide, urocortin (Ucn) [35], to produce cAMP with similar half-maximally effective concentrations (EC50) [6], [9], [35]. Cells producing CRF2 receptor generate cAMP especially in response to Svg and Ucn [14], [19], [20], as indicated by low EC50 values and high affinity binding of these peptides. The 38 residue peptides urocortin II (Ucn II) and human Ucn-related peptide (hURP) have been recently identified as CRF2 receptor-specific ligands [13], [17], [23]. They were found to exhibit high affinity binding to CRF2 receptor and activate cAMP production only in cells containing this receptor subtype.
Several studies on the structure-activity relationship (SAR) of CRF have been carried out [2], [4], [15], [24], [25]. It was observed that N-terminal deletion of residues 1–8 of the agonist sequence generally lowers the affinity for CRF1 receptor and generates CRF receptor antagonists [25]. Moreover, the introduction of a lactam bridge between residues 30 and 33 of the CRF sequence results in a significant increase of binding affinity for either CRF receptor subtype [12], [26], [27]. It was therefore speculated that the lactam ring may promote conformational stability, which may originally have been induced by the N-terminus of CRF [12].
In view of the existence of CRF receptor subtypes and their different biological functions (reviewed in [7], [22]), it is important to determine the structural properties of the peptidic ligands that are crucial for receptor selectivity. The only CRF2 receptor-selective antagonist developed to date is the 30 residue peptide antisauvagine-30 (aSvg), which represents a truncated Svg with additionally modified N-terminus [27]. Taking into consideration that aSvg is a CRF2 receptor-selective antagonist derived from an unselective agonist, we decided to determine the importance of the N-terminal domain of CRF-like peptides for discrimination between receptor subtypes (Table 1).
Therefore, we synthesized analogs on the basis of the structures of human CRF12–41 (hCRF12–41), human URP12–38 (hURP12–38) and [d-Phe11,His12]Svg11–40 (aSvg) (Fig. 1). We prolonged these peptides N-terminally by consecutive addition of one or two amino acids (Table 1) and tested the resulting compounds for their binding affinities to rat CRF1 (rCRF1) and murine CRF2β (mCRF2β) receptor produced by transfected HEK 293 cells. Additionally, these compounds were tested for their capability to stimulate intracellular cAMP production. These experiments were performed with the objective to identify the residues of these peptides, which are crucial for their biological potency and intrinsic activity (efficacy).
Section snippets
Peptide synthesis
Fully protected peptides were synthesized following the Fmoc strategy (0.1 mmol scale) on TentaGel S RAM™ resin (Rapp) with an ABI 433A peptide synthesizer (Applied Biosystems). The peptides were cleaved from the resin under standard conditions [3] and purified by reversed-phase HPLC on Vydac C18 columns ( mm, 5 μm particle size, 300 Å pore size; Vydac, Hesperia, CA). Mass spectra of purified peptides were recorded with a plasma desorption mass spectrometer BioIon 20 (Applied Biosystems,
Binding properties of hCRF-, hURP- and aSvg-analogs
Competition binding experiments were performed using the radioligands -[Tyr0]oCRF for rCRF1 receptor and -[Tyr0]Svg for mCRF2β receptor. N-terminal truncation of oCRF by 12 residues resulted in a significant loss of affinity to either receptor subtype as indicated by Ki values >1 μM with oCRF13–41 as ligand (data not shown). Thus, we concluded that residues 13–41 of CRF were not sufficient for high affinity binding to either CRF receptor subtype. The analogue hCRF3–41 exhibited high
Discussion
The objective of this SAR study was to investigate the influence of the N-terminal domain of CRF and CRF-like peptides on their selectivity between the two major subtypes of CRF receptors characterized to date.
We confirmed that, in contrast to hCRF, which preferentially binds to CRF1 receptors [14], [19], [20], hURP exhibited specific high affinity binding to CRF2 receptors [23]. From the observation that full length hURP exhibited a similar efficacy like hCRF to activate cAMP production by
Acknowledgements
We gratefully acknowledge Thomas Liepold for excellent technical assistance and Drs. Thomas Zeyda and Klaus Eckart for helpful discussions. We thank Drs. M.G. Rosenfeld and C.R. Lin (Eucaryotic Regulatory Program, School of Medicine, UCSD, La Jolla, CA) for kindly providing us with the HEK-mCRF2β cells.
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