Introduction

The 41-amino peptide corticotropin-releasing factor (CRF) was first isolated from ovine hypothalamus [49]. Initially, the action of CRF appeared to be restricted to regulating ACTH secretion by the pituitary [17-18,43,49]. The discovery that CRF-expressing neurons existed in pathways outside of the hypothalamus, however, led to the realization that CRF's function extended far beyond the classical action of a hormone in the hypothalamic-pituitary adrenocortical axis [2,12,15,20,46,50,53]. It then became apparent that brain CRF neurotransmission was critical for behavioral, neuroendocrine, and autonomic responses to stress. In fact, CRF-induced activation of brain CRF1 receptors is both necessary and sufficient to define the stress response.

Urocortin 1, a second mammalian CRF-like peptide was identified following the sequencing of two novel CRF-like peptides urotensin I [25] and sauvagine [31] in fish and amphibians [34,45]. Subsequently, human, sheep, rat, and mouse urocortin 1 were shown to have 40-amino acid sequences [4,16,51,55] with high degrees of homology with fish urotensin I. While urocortin 1 is discretely expressed in the central nervous system this CRF-like peptide is widely distributed in the periphery [3,22].

In 2001, two novel urocortin-like peptides, urocortin 2 and urocortin 3 [26,42], were cloned 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) [21](see Table 1). The remarkably discrete expression of urocortin 2 (stresscopin-related peptide) and urocortin 3 (stresscopin) in the central nervous system does not conform with the broad distribution of CRF pathways and the discrete pattern of urocortin 1 pathways [21,26,42]. Furthermore, the central distribution of urocortins 1-3 and CRF2 receptor-expressing neurons suggests that all three peptides may serve as major CRF2 receptor ligands in highly localized and specific manner. High expression of urocortin 3 mRNA in the lateral septum and urocortin 2 mRNA in the locus coeruleus is particularly intriguing with regard to the possibility that CRF2 receptor-mediated mechanisms may modulate and oppose in certain situations neural stress responses evoked by CRF1 receptor activation. In the periphery, urocortin 2 mRNA is detected in the heart, adrenal gland, and peripheral blood cells [21,42]. The highest peripheral levels of urocortin 3 mRNA expression have been detected in the colon, pancreatic islet cells, muscle, adrenal gland, and skin [21,26]. Because urocortin 2 and urocortin 3 were discovered by molecular cloning strategies [21,26,42], their exact size has not been established yet. The structure of both precursor genes appears to predict 38-amino acid mature peptides [26,42] although one group [21] postulated the existence of N-terminally extended peptides 40 (urocortin 3) and 43 (urocortin 2) amino acids in length (Table 1). The final forms of post-translationally modified urocortin 2 and urocortin 3 peptides in brain and peripheral tissues remains to be determined. However, because the human and mouse homologues of both precursors are less conserved in the extended N-terminus than in the remaining sequence (Table 1), it seems more likely that urocortin 2 and urocortin 3 exist as 38 amino acid peptides. Like CRF 1 and urocortin 1 [51], urocortin 2 and urocortin 3 possess biological activity only when they are C-terminally amidated [21]. Only four amino acids are completely conserved among CRF peptides (Table 1). Therefore, secondary structure rather than linear sequence homology most likely determines differences in biological activity of the members of the CRF peptide family [10,19,21,26,42].

The CRF₁ receptor

The CRF1 receptor, a 415-446 amino acid polypeptide, has been cloned from a variety of species ranging from fish to man [1,6-8,32,35,37-38,40,52,54]. The CRF₁ receptor is widely expressed in the central nervous system with high levels of expression in cortex, cerebellum, hippocampus, basolateral amygdala, bed nucleus of thes stria terminalis, olfactory bulb and anterior pituitary [5,37,41]. In the periphery, CRF₁ receptor mRNA is expressed at low levels in the skin, ovary, testis and adrenal gland [33,37,52].

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 [8,13,16,39]. In contrast, urocortin 2 and urocortin 3 do not bind to or activate CRF₁ receptors [9,21,26,42]. Therefore, CRF and urocortin 1 can be classified as the endogenous ligands for mammalian CRF₁ receptors.

The CRF₂ receptor

Complementary DNAs for the CRF₂ receptor have also been isolated from large number of vertebrate species [1,8,23-24,26,28,30,36,40,44,48]. Three functional splice variants [9,28,48] have been identified for the mammalian CRF₂ receptor. The CRF(2a) receptor variant is only expressed in non-mammalian species [1,8,36,40], while the 430-438 amino-acid CRF_(2b) receptor and the CRF_(2a) receptor are both expressed in mammals [9,23-24,26,28,30,48]. Expression of the 397-amino acid CRF_(2c) receptor has only been detected in limbic regions of the human central nervous system [48]. 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 ñ50 kb in size and contains 15 exons [14]. The first four exons give rise to the different 5'-ends of the splice variants hCRF2(a), hCRF2(b) and hCRF_2(c), respectively; exons 5-15 form the common parts of the various hCRF₂ splice variants. Exons 1 and 2, which are separated by ñ10.5 kb intronic sequence encode the CRF_2(b)-specific part, followed by the CRF_2(c) and CRF_2(a) exons [14].

In rodents, CRF_(2a) receptor mRNA is expressed primarily in brain neurons while CRF_(2b) receptor mRNA is detected in non-neuronal brain structures and peripheral tissues [11]. The CRF_(2a) receptor is the dominant CRF₂ receptor splice variant expressed in the mammalian brain and is mainly found in areas that are critically involved in the mediation of stress responses [3,29]. In humans and tree shrews, CRF_(2a) receptor mRNA expression is broader than in rodent brain [24,27]. Because CRF_(2b) receptor mRNA can be detected in neuronal structures 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 CRF_2(a) receptor is the major splice variant found in the peripheral tissues of humans [28,48], while the CRF_2(b) splice variant is the CRF₂ receptor peripherally expressed in rodents. The main expression sites for the rodent CRF_(2b) receptor have been reported for heart, lung, skeletal muscle, gastrointestinal tract, testis and ovaries [11,24].

Pharmacological characterization of the CRF₂ receptor splice variants revealed no major differences between CRF_(2a), CRF_(2b) and CRF_(2c) receptors [16,24,47-48]. However, the binding profiles of these three CRF₂ receptors strongly diverge from the binding profile of the CRF₁ receptor [13,16,21,26,39,42]. The non-mammalian 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. In agreement with the binding data, a similar rank order of potency is typically observed when stimulation of intracellular cyclic AMP accumulation is measured [13,16,21,26,42]. Therefore, current evidence suggests that urocortin 2 and urocortin 3 represent the endogenous ligands for mammalian CRF₂ receptor variants. Urocortin 1 is an endogenous ligand with equal potencies in activating CRF₁ and CRF₂ receptors.

Unsolved issues

Recently a third CRF receptor, termed CRF₃, was cloned from catfish [1]. The catfish CRF[18] receptor was not detected in salmon [40]. 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 [1]. 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 [10]). Therefore, human urocortin 2 may not be processed into a biologically active peptide in vivo [21,26]. The isolation of the urocortin 2 peptide from native human tissues would resolve this issue. In literature, the abbreviation UCN is frequently used for urocortins. Because the abbreviation CRF exists a similar abbreviation for the urocortins is currently discussed.

Table 1: Human, rat and mouse versions of CRF, urocortin 1, -2 and -3 are compared with human stresscopin, stresscopin-related peptide (extended versions of human urocortin 3 and -2) and fish urotensin I and frog sauvagine. The amino acids highlighted are conserved among all peptides.

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