Department of Psychiatry and Veterans Affairs Healthcare System,
University of California San Diego, La Jolla, California (R.L.H.);
Neurocrine Biosciences, Inc., San Diego, California (D.E.G.);
Department of Physiology, University of California San Francisco,
California (M.F.D.); Stress Neurobiology Laboratory, Department of
Psychiatry and Behavioral Sciences, Emory University, Atlanta, Georgia
(P.M.P.); Clayton Foundation Laboratories for Peptide Biology and
Laboratory of Neuronal Structure and Function, Salk Institute for
Biological Studies, La Jolla, California (W.W.V.); c./o. Axovan. Ltd.,
Allschwil-Switzerland (F.M.D.)
Receptors for corticotropin-releasing factor (CRF) are members of
a family of G protein-coupled receptors ("Family B") that respond
to a variety of structurally dissimilar releasing factors, neuropeptides, and hormones (including secretin, growth
hormone-releasing factor, calcitonin, parathyroid hormone, pituitary
adenylate cyclase-activating polypeptide, and vasoactive intestinal
polypeptide) and signal through the cyclic AMP and/or calcium pathways.
To date, three genes encoding additional CRF-like peptides (urocortins)
have been identified in mammals. The urocortins and CRF bind with
differential ligand selectivity at the two mammalian CRF receptors.
This report was prepared by the International Union of Pharmacology
Subcommittee on CRF Receptors, to summarize the current state of CRF
receptor biology and to propose changes in the classification and
nomenclature of CRF ligands and receptors.
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I. Introduction |
In 1981, the 41-amino acid corticotropin-releasing
factor (CRF1)
peptide was isolated from ovine hypothalamus (Vale et al., 1981
). Initially, the action of CRF appeared to be restricted to regulating ACTH secretion by pituitary corticotrope cells (Vale et al., 1981; Dunn
and Berridge 1990; Hauger and Dautzenberg, 1999; Smagin et al., 2001).
The central distribution of CRF pathways, however, suggested that
CRF's function extended far beyond the classical action of a hormone
(Swanson et al., 1983; De Souza et al., 1984; Wynn et al., 1984; Vale
et al., 1997; Arborelius et al., 1999; Heinrichs and De Souza 1999;
Dautzenberg et al., 2001a). Because CRF was shown to generate
neuroendocrine, autonomic, and behavioral stress responses, it was
hypothesized that CRF contributed to the development of stress and
affective disorders by over-activating its receptors expressed in
various brain neurons in neocortex, the amygdala and its extended
neurocircuits, and brainstem nuclei.
Urocortin, a second mammalian CRF-like peptide was identified after two
novel CRF-like peptides, urotensin I (Lederis et al., 1982) and
sauvagine (Montecucchi and Henschen, 1981), were discovered in fish and
amphibian species in addition to CRF (Okawara et al., 1988;
Stenzel-Poore et al., 1992). Human, sheep, rat, and mouse urocortin
possess a 40-amino acid sequence (Vaughan et al., 1995; Donaldson et
al., 1996; Zhao et al., 1998; Cepoi et al., 1999) and a high degree of
homology with fish urotensin I. Although the Edinger-Westphal locus,
the hypothalamus, and a small population of forebrain neurons express
substantial levels of urocortin (Bittencourt et al., 1999), urocortin
is more broadly expressed in the periphery, especially in the
pituitary, gastrointestinal tract, testis, cardiac myocytes, thymus,
spleen, and kidney (Kageyama et al., 1999).
Very recently, two novel isoforms of urocortin, urocortin 2 and
urocortin 3 (Lewis et al., 2001; Reyes et al., 2001), were cloned at
the Salk Institute from human and mouse cDNA libraries. At the same
time, another group identified two similar peptides, which they named
stresscopin (which is homologous with urocortin 3) and
stresscopin-related peptide (which is homologous with urocortin 2) (Hsu
and Hsueh, 2001). Urocortin 2 (stresscopin-related peptide) and
urocortin 3 (stresscopin) are discretely expressed in the central
nervous system with a pattern distinct from the known CRF and urocortin
pathways (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001).
Furthermore, the central distribution of urocortin 1-, urocortin 3-, and CRF2 receptor-expressing neurons suggests
that urocortin 1 may serve as the major CRF2
receptor ligand in the hindbrain whereas urocortin 3 may serve as the
major CRF2 receptor ligand in the forebrain.
Urocortin 2 or a novel endogenous ligand may signal at
CRF2 receptors expressed in certain brain regions
lacking urocortin 1 or urocortin 3 innervations (e.g., the hippocampus
and certain regions of the cerebral cortex). Because neither
CRF1 nor CRF2 receptors
have yet been identified in the locus coeruleus, central nucleus of the
amygdala, and several other stress-sensitive brain structures, it is
possible that a novel CRF receptor may eventually be cloned (Hsu and
Hsueh, 2001; Li et al., 2002). In the periphery, urocortin 2 mRNA is
detected in the heart, adrenal gland, and peripheral blood cells (Hsu
and Hsueh, 2001; Reyes et al., 2001). The highest peripheral levels of
urocortin 3 mRNA expression have been detected in the gastrointestinal tract, muscle, adrenal gland, and skin (Hsu and Hsueh, 2001; Lewis et
al., 2001). Because urocortin 2 and urocortin 3 have been identified by
molecular cloning strategies (Hsu and Hsueh, 2001; Lewis et al., 2001;
Reyes et al., 2001), their exact size has not been established yet. The
structure of both precursor genes appears to predict 38-amino acid
mature peptides (Lewis et al., 2001; Reyes et al., 2001) although one
group (Hsu and Hsueh, 2001) postulated the existence of N-terminally
extended peptides 40 (urocortin 3) and 43 (urocortin 2) amino acids in
length. However, because the human and mouse homologues of both
precursors are less conserved in the extended N terminus than in the
remaining sequence, it seems more likely that urocortin 2 and urocortin
3 exist as 38-amino acid peptides. Furthermore, the 38- and the
40-/43-amino acid versions of both ligands possess similar
pharmacological potencies. Because CRF ligands rapidly lose agonistic
potency upon N-terminal truncation (Rivier et al., 1984; Brauns et al.,
2002) the pharmacological data also speak in favor of the 38-amino acid
variants for urocortin 2 and urocortin 3. This issue will not be
conclusively resolved until endogenous forms of these two urocortin
peptides are isolated in various species. Like CRF (Vale et al., 1981)
and urocortin 1 (Vaughan et al., 1995), urocortin 2 and urocortin 3 possess biological activity only when they are C-terminally amidated
(Hsu and Hsueh, 2001). Only 4 amino acids are completely conserved among CRF peptides (Fig. 1). Therefore,
secondary structure rather than linear sequence homology most likely
determines differences in biological activity of the members of the CRF
peptide family (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al.,
2001; Dautzenberg and Hauger, 2002).
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Fig. 1.
Alignment of the members of the CRF peptide family.
The amino acids that are homologous between the CRF peptides are boxed.
h, human; m, mouse; o, ovine; UCN 1, urocortin 1; UCN 2, urocortin 2;
UCN 3, urocortin 3.
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II. The Corticotropin-Releasing Factor Type 1 Receptor |
The CRF1 receptor, a 415-446 amino acid
polypeptide, has been cloned from a variety of species including man
(Chen et al., 1993; Vita et al., 1993), tree shrew (Palchaudhuri et
al., 1998), mouse (Vita et al., 1993), rat (Chang et al., 1993; Perrin
et al., 1993), sheep (Myers et al., 1998), chicken (Yu et al., 1996), frog (Dautzenberg et al., 1997), and fish (Arai et al., 2001; Pohl et al., 2001). Although a larger number of splice variants of the
CRF1 receptor cDNA have been identified (Chang et
al., 1993; Chen et al., 1993; Ross et al., 1994; Myers et al., 1998; Grammatopoulos et al., 1999) (Fig. 2),
they have not been shown to encode functional receptors in vivo due to
their low binding affinity or lack of activation in recombinant systems
(reviewed in Dautzenberg et al., 2001a). The CRF1
receptor is abundantly expressed in the central nervous system with
major expression sites in cortex, cerebellum, hippocampus, amygdala,
olfactory bulb, and pituitary (Potter et al., 1994; Chalmers et al.,
1996; Palchaudhuri et al., 1998). In the periphery,
CRF1 receptor mRNA is expressed at low levels in
the skin, ovary, testis, and adrenal gland (Vita et al., 1993; Nappi
and Rivest, 1995; Palchaudhuri et al., 1998).
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Fig. 2.
Two-dimensional structure of the human
CRF1 and CRF2 receptors. Identical amino acids
between both receptors are represented as filled circles whereas
divergent residues are shown as open circles. The arrows indicate sites
for insertion or deletion of exons in nonfunctional variants of the
CRF1 and CRF2 receptor (Chen et al., 1993;
Myers et al., 1998; Grammatopoulos et al., 1999; Miyata et al., 1999).
The symbols { and } indicate the deletion of a 40-amino acid exon
in a nonfunctional splice variant of the human CRF1
receptor (Ross et al., 1994) whereas the symbol ] represents the
common splice site for the three CRF2 variants
CRF2(a), CRF2(b), and CRF2(c).
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Binding and functional studies using cell lines recombinantly or
endogenously expressing CRF1 receptors revealed a
distinct ligand-selective profile whereby human and ovine CRF,
urocortin 1, urotensin I, and sauvagine all bind with high affinity to
the mammalian CRF1 receptor and activate the
cyclic AMP signaling pathway (Donaldson et al., 1996; Dautzenberg et
al., 1997, 2001b; Perrin et al., 1999). In contrast, urocortin 2 and
urocortin 3 do not bind to or activate CRF1
receptors (Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al.,
2001). Therefore, CRF and urocortin 1 can be classified as the
endogenous ligands for mammalian CRF1 receptors
(Fig. 3).
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Fig. 3.
Pharmacological properties of the four mammalian
CRF agonists CRF, urocortin 1, urocortin 2, and urocortin 3 at the
human CRF1 and CRF2 receptors. Binding of the
four natural agonists to the CRF1 (A) and CRF2
(B) receptors reveals a distinct profile for each ligand with urocortin
1 being a high-affinity ligand to both receptors and CRF being a high
affinity ligand to the CRF1 and a low-affinity ligand to
the CRF2 receptor. Urocortin 2 and urocortin 3, however,
only bind to the CRF2 receptor. Similar findings are
observed in cAMP stimulation studies with recombinant receptors (C and
D). In contrast to the binding studies, however, a very weak agonistic
potency (>1 µM) is observed for urocortin 2 and urocortin 3 at the
CRF1 receptor (C).
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III. The Corticotropin-Releasing Factor Type 2 Receptor |
Complementary DNAs for the CRF2 receptor
have been isolated from man (Liaw et al., 1996; Valdenaire et al.,
1997; Kostich et al., 1998), tree shrew (Palchaudhuri et al., 1999),
rat (Lovenberg et al., 1995b), mouse (Kishimoto et al., 1995; Perrin et
al., 1995; Stenzel et al., 1995), frog (Dautzenberg et al., 1997), and
fish (Arai et al., 2001; Pohl et al., 2001). Three functional splice
variants (Lovenberg et al., 1995b; Kostich et al., 1998) and a
truncation variant (Miyata et al., 1999) have been identified for the
mammalian CRF2 receptor. The
CRF2(a) receptor variant is only expressed
in nonmammalian species (Dautzenberg et al., 1997; Arai et al., 2001;
Pohl et al., 2001), whereas the 430-438 amino acid
CRF2(b) receptor and the
CRF2(a) receptor are both expressed in mammals
(Kishimoto et al., 1995; Lovenberg et al., 1995b; Perrin et al., 1995;
Stenzel et al., 1995; Valdenaire et al., 1997; Palchaudhuri et al.,
1999). Expression of the 397-amino acid CRF2(c)
receptor has only been detected in limbic regions of the human central
nervous system (Kostich et al., 1998). Splicing of the
CRF2 receptor variants occurs at the extreme 5'
terminus of the receptor gene and reflects usage of different promoters in man. The hCRF2 gene, which is located on
chromosome 7p14-15, is ~50 kb in size and contains 15 exons
(Dautzenberg et al., 2000). The first four exons give rise to the
different 5'-ends of the splice variants
hCRF2(a), hCRF2(b) and
hCRF2(c), respectively; exons 5 to 15 form the
common parts of the various hCRF2 splice variants. Exons 1 and 2, which are separated by ~10.5 kb intronic sequence encode the CRF2(b)-specific part,
followed by the CRF2(c) and
CRF2(a) exons (Dautzenberg et al., 2000).
In rodents, CRF2(a) receptor mRNA is expressed
primarily in brain neurons whereas CRF2(b)
receptor mRNA is detected in non-neuronal brain structures such
as the choroid plexus cerebral arterioles and peripheral tissues
(Lovenberg et al., 1995a). The CRF2(a) receptor
is the dominant CRF2 receptor splice variant
expressed in the mammalian brain. Central CRF2(a)
receptors are expressed in a discrete pattern with highest densities in
the lateral septum, ventromedial hypothalamus, cortical nucleus of the
amygdala, dorsal raphe, nucleus of the solitary tract, and the choroid
plexus; whereas, a widespread but discrete expression of
CRF1 receptors is found in neocortex and the
amygdala and its extended neurocircuits (Bittencourt et al., 1999; Li
et al., 2002). In humans and tree shrews, CRF2(a)
receptor mRNA has also been detected in the brainstem, olfactory bulbs,
cortex, cerebellum, retina, and pituitary (Palchaudhuri et al., 1999;
Sanchez et al., 1999). Because CRF2(b) receptor mRNA can be detected in neuronal structures such as retina and cerebellum, the distribution of these two splice variants may overlap
in primates and primate-like animals. In the periphery, substantial
expression of CRF2 receptor can be found in the
heart, skeletal muscle, vasculature, and gastrointestinal tract. The CRF2(a) receptor is the major splice variant
found in the peripheral tissues of humans (e.g., heart and skeletal
muscle) (Valdenaire et al., 1997; Kostich et al., 1998), whereas the
CRF2(b) splice variant is the
CRF2 receptor peripherally expressed in rodents. The main expression sites for the rodent CRF2(b)
receptor have been reported for heart, lung, skeletal muscle,
gastrointestinal tract, testis, and ovaries (Lovenberg et al., 1995a;
Palchaudhuri et al., 1999).
Pharmacological characterization of the CRF2
receptor splice variants revealed no major differences between
CRF2(a), CRF2(b), and
CRF2(c) receptors (Donaldson et al., 1996;
Kostich et al., 1998; Palchaudhuri et al., 1999). However, the binding
profiles of these three CRF2 receptors strongly
diverge from the binding profile of the CRF1
receptor (Donaldson et al., 1996; Perrin et al., 1999; Dautzenberg et
al., 2001b; Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al.,
2001). The nonmammalian CRF peptides urotensin I and sauvagine and the
mammalian peptides urocortin 1, urocortin 2, and urocortin 3 generally
bind with up to 100-fold higher affinities to the
CRF2 receptor than species homologues of CRF
(Fig. 3B). In agreement with the binding data, a similar rank order of
potency is typically observed when stimulation of intracellular cyclic
AMP accumulation is measured (Donaldson et al., 1996; Dautzenberg et
al., 2001b; Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al.,
2001) (Fig. 3D). Therefore, currently, urocortin 2 and urocortin 3 are
generally considered to represent the endogenous ligands for mammalian
CRF2 receptor variants, whereas urocortin 1 is
thought to be an endogenous ligand for both the CRF1 and CRF2 receptors.
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IV. Proposed Nomenclature |
Early clinical neuroendocrine studies, particularly those directed
at investigating the role of CRF in regulating the
hypothalamic-pituitary-adrenal axis, defined this peptide's function
to be a stimulator of ACTH secretion. Accordingly, the term
"corticotropin-releasing hormone" gained acceptance in the
neuroendocrine literature. However, ample evidence has accumulated over
the past two decades demonstrating that CRF's physiological function
extends far beyond the biology of a hormone. In fact, CRF most likely
plays a role in immune, neurodegenerative, neuropsychiatric, and
gastrointestinal disorders. Thus, the term "corticotropin-releasing
factor" more appropriately designates the function of this peptide.
It is recommended that CRF rather than corticotropin-releasing hormone
(CRH) should be used in keeping with the nomenclature for other
important factors (i.e., transforming growth factor and
brain-derived neurotrophic factor) that exhibit a multiplicity of
biological actions.
The three-dimensional structure, CRF2 receptor
binding profile, and biological actions of urocortin 2 and urocortin 3 are similar to each other and to urocortin 1 (see Hsu and Hsueh, 2001; Lewis et al., 2001; Reyes et al., 2001; Dautzenberg and Hauger, 2002).
These novel ligands should therefore retain the following designations:
urocortin 2 and urocortin 3. Stresscopin, which has recently been used
to designate the peptide with urocortin 2's sequence, implies its role
is to mediate an organism's "coping" with stress. Activation of
central CRF2 receptors can produce anxiogenesis
or anxiolysis depending on the brain site (Reul and Holsboer, 2002).
Thus, the term "stresscopin" (Hsu and Hsueh, 2001) only covers one
aspect of the behavioral response mediated by brain
CRF2 receptor signaling. Therefore, the urocortin
2 and urocortin 3 should be used in place of the terms stresscopin and stresscopin-related peptide, respectively. Finally, since the urocortin
family contains three members, urocortin 1 will replace the earlier
generic term "urocortin". Table 1
summarizes all of the recommended changes for the nomenclature of CRF
peptides.
A revised nomenclature for CRF receptors is summarized in Table
2. Although several alternative
terminologies such as CRH1, CRFR1 or CRF-R1 have been used to designate
the CRF1 receptor, the term
CRF1 receptor should be utilized. Because the
CRF2 receptor is highly selective for urocortin 1 over CRF, renaming the CRF2 receptor as the
urocortin receptor was considered. However, urocortin 1 binds equally
well to the CRF1 and CRF2
receptors. Therefore, redesignating the CRF2
receptor as the urocortin receptor fails to distinguish the biology of
the two receptors. Therefore, the term CRF2
receptor should be used for the time being to maintain clarity (Table
2). As new insight into the normal and pathophysiological actions of
the CRF2 receptor subtypes is gained, and as
species variations and the behavioral roles of the
CRF2 receptor subtypes become understood, the
naming of this CRF receptor may require future revisions. In addition,
in accordance with IUPHAR convention, CRF receptor splice variants
should be listed alphabetically using Roman rather than Greek letters
as have appeared in the current literature (Humphrey and Barnard,
1998). Thus, the three CRF2 receptor
splice variants will be designated as CRF2(a),
CRF2(b), and CRF2(c).
Finally, as novel CRF1 and
CRF2 splice variants become identified, they will
only be classified as formal CRF receptors if they encode
physiologically functional receptor proteins but not simply polymerase
chain reaction products. Numerous low-affinity or nonfunctional CRF
receptor variants possess an incomplete structure due to either exon
deletions or insertions, which mostly occur in a single species
(reviewed in Dautzenberg et al., 2001a).
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V. Unresolved Issues |
Recently a third CRF receptor, termed CRF3,
was cloned from catfish (Arai et al., 2001). The catfish
CRF3 receptor was not detected in salmon (Pohl et
al., 2001). The catfish CRF1 and
CRF3 receptors are highly homologous.
Interestingly, CRF3 receptor expression is
restricted to the catfish pituitary, a tissue that normally expresses
CRF1 receptors in other species (Arai et al., 2001). Species homologues for this receptor may not exist.
Human urocortin 2 lacks the standard consensus site required for
proteolytic cleavage and C-terminal amidation, which is a prerequisite
for biological potency (reviewed in Dautzenberg and Hauger, 2002).
Therefore, human urocortin 2 may not be processed into a biologically
active peptide in vivo (Hsu and Hsueh, 2001; Reyes et al., 2001). The
isolation of the urocortin 2 peptide from native human tissues would
resolve this issue.
We thank Profs. A. J. Harmar and M. Spedding for liaison with the IUPHAR Committee on Receptor Nomenclature
and Drug Classification (NC-IUPHAR).
Address correspondence to: Dr. Frank M. Dautzenberg, c./o.
Axovan Ltd., Gewerbestrasse 16, CH-4070 Allschwil, Switzerland. E-mail:
frank.dautzenberg{at}t-online.de
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receptors and the role of position 185 for receptor-ligand selectivity.
Neuropharmacology
39:
1368-1376[CrossRef][Medline].
and CRF2
receptor mRNAs are differentially distributed between the rat central nervous system and peripheral tissues.
Endocrinology
136:
4139-4142[Abstract].
