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Peptides of the corticotropin-releasing factor (CRF) family are expressed throughout the central nervous system (CNS) and in peripheral tissues where they play diverse roles in physiology, behavior, and development. Current data supports the existence of four paralogous genes in vertebrates that encode CRF, urocortin/urotensin 1, urocortin 2 or urocortin 3. Corticotropin-releasing factor is the major hypophysiotropin for adrenocorticotropin, and also functions as a thyrotropin-releasing factor in non-mammalian species. In the CNS, CRF peptides function as neurotransmitters/neuromodulators. Recent work shows that CRF peptides are also expressed at diverse sites outside of the CNS in mammals, and we found widespread expression of CRF and urocortins, CRF receptors and CRF binding protein (CRF-BP) genes in the frog Xenopus laevis. The functions of CRF peptides expressed in the periphery in non-mammalian species are largely unexplored. We recently found that CRF acts as a cytoprotective agent in the X. laevis tadpoletail, and that the CRF-BP can block CRF action and hasten tailmuscle cell death. The expression of the CRF-BP is strongly upregulated in the tadpoletail at metamorphic climax where it may neutralize CRF bioactivity, thus promoting tail resorption. Corticotropin-releasing factor and urocortins are also known to be cytoprotective in mammalian cells. Thus, CRF peptides may play diverse roles in physiology and development, and these functions likely arose early in vertebrate evolution.
Fig. 1. Schematic representation of relative binding aYnities and speciWcities of: (A) mammalian and (B) X. laevis corticotropin-releasing factor-like peptides,
their receptors (CRF1 and CRF2), and the CRF binding protein (CRF-BP). The question mark indicates that a urocortin 2 gene has not yet been
identiWed in X. laevis. Solid lines with arrows indicate high aYnity, while dashed lines with arrows indicate lower aYnity binding.
Fig. 2. Tissue distribution of mRNAs for corticotropin-releasing factor system components in juvenile Xenopus laevis tissues analyzed by RT-PCR. RNA
isolation and RT-PCR is described by Boorse et al. (2005a) and the PCR primer sequences are given in Table 1. (A) Expression analysis in brain and pituitary.
(B) Expression analysis in diverse peripheral tissues. Corticotropin-releasing factor (CRF), urocortin 1 (UCN1), urocortin 3 (UCN3), CRF binding
protein (CRF-BP), CRF receptor 1 (CRF1) and CRF receptor 2 (CRF2). Ribosomal L8 (rpL8) was used as a housekeeping gene to control for RNA quality
and loading. Shown are representative ethidium bromide-stained gels of RT-PCR products; RNA was harvested from tissues of three females and similar
results were obtained with each. PCR reactions conducted on reverse transcription reactions in which the reverse transcriptase was omitted (minus RT
reactions) produced no bands, thus conWrming the absence of genomic DNA contamination + and ¡ on the Wgure. RT-PCR reactions conducted on RNA
isolated from whole blood or packed blood cells produced no bands, which shows that the positive results that we obtained in other tissues are not due to
contamination from blood cells. However, we cannot rule out the possibility that some blood cell types express CRF system signaling components but
their abundance was below the level of detection of our assay.
Fig. 3. Detection of CRF and CRF-BP proteins in frog tissue extracts by
radioimmunoassay and crosslinking assay, respectively. (A) To determine
if mRNA expression correlates with protein we used a homologous radioimmunoassay
for X. laevis CRF (Boorse and Denver, 2004b) to analyze
CRF-immunoreactivity (CRF-ir) in frog tissue extracts. The graph shows
the relative displacement of [125I]-Xenopus laevis CRF (xCRF) by increasing
concentrations of radioinert CRF (closed squares) or dilutions of tissue
extracts (closed circlesD skin extract; open circlesDheart extract;
open diamondsD liver extract). Each tissue was acetic-acid extracted then
analyzed for CRF peptide content by speciWc radioimmunoassay as
described (Boorse and Denver, 2004b). Serial dilutions of extracts from
the skin or heart produced displacement curves that were parallel to the
frog CRF (xCRF) standard. The CRF peptide content of skin was
0.303 § 0.004 pg CRF/mg tissue and heart 0.508§ 0.126 pg CRF/mg tissue
(mean §SEM; n D3); liver was nondetectable. These values are considerably
lower than peptide content in the brain (121.63§ 13.44 pg CRF/mg
tissue; mean§ SEM; n D 5) but well within the limits of detection of our
assay (Boorse and Denver, 2004b). We were unable to detect CRF-ir in
other tissues that expressed CRF mRNA (data not shown). (B) We used a
chemical crosslinking assay with [125I]-CRF (Valverde et al., 2001) to
detect CRF-BP in tissue extracts (brain, heart, lung, kidney, intestine,
stomach, and liver was analyzed.) We detected the CRF-BP in brain and
intestine but not other tissues (data not shown). For brain extracts we
used 30 g while for intestine we used 75 g of total protein. SpeciWc binding
of crosslinked proteins was shown by the addition of excess (1 M)
radioinert xCRF prior to crosslinking and fractionation by 10% SDS–
PAGE. Data shown are representative of results obtained with tissues isolated
from three female frogs.
