Urocortin II: a member of the corticotropin-releasing factor (CRF) neuropeptide family that is selectively bound by type 2 CRF receptors

Abstract

Here we describe the cloning and initial characterization of a previously unidentified CRF-related neuropeptide, urocortin II (Ucn II). Searches of the public human genome database identified a region with significant sequence homology to the CRF neuropeptide family. By using homologous primers deduced from the human sequence, a mouse cDNA was isolated from whole brain poly(A)(+) RNA that encodes a predicted 38-aa peptide, structurally related to the other known mammalian family members, CRF and Ucn. Ucn II binds selectively to the type 2 CRF receptor (CRF-R2), with no appreciable activity on CRF-R1. Transcripts encoding Ucn II are expressed in discrete regions of the rodent central nervous system, including stress-related cell groups in the hypothalamus (paraventricular and arcuate nuclei) and brainstem (locus coeruleus). Central administration of 1-10 microg of peptide elicits activational responses (Fos induction) preferentially within a core circuitry subserving autonomic and neuroendocrine regulation, but whose overall pattern does not broadly mimic the CRF-R2 distribution. Behaviorally, central Ucn II attenuates nighttime feeding, with a time course distinct from that seen in response to CRF. In contrast to CRF, however, central Ucn II failed to increase gross motor activity. These findings identify Ucn II as a new member of the CRF family of neuropeptides, which is expressed centrally and binds selectively to CRF-R2. Initial functional studies are consistent with Ucn II involvement in central autonomic and appetitive control, but not in generalized behavioral activation.

Figure 1

(A) Predicted amino acid sequence of murine Ucn II. The start methionine, marked in bold, is located upstream of the peptide coding region, which is boxed. The complete nucleotide sequence has been deposited in GenBank (accession no. AF331517). (B) Alignment of mouse Ucn II with homologous human and fish peptides (URPs) and with rat Ucn and rat/human CRF; residues identical to the mouse Ucn II sequence are boxed. ■, Amidation site (putative for human URP).

Figure 2

Ucn II mRNA expression in the rat brain. Darkfield photomicrographs showing labeling (white grains) observed over select regions using an isotopically labeled antisense cRNA probe generated from a mouse Ucn II cDNA. Positive hybridization signals are seen over the paraventricular nucleus of the hypothalamus (A), principally over its magnocellular division (pm), with more diffuse signal seen over the parvocellular aspect (mp), and broadly over the locus coeruleus (LC; B), facial motor nucleus (VII, C) and meninges (men) at the ventral surface of the brain. CBL, cerebellum; v3, third ventricle; v4, fourth ventricle. [Magnifications: ×75 (A and B); ×50 (C).]

Figure 3

Cellular activation patterns in response to central Ucn II microinjection. (A-C and E) Brightfield photomicrographs of immunoperoxidase preparations showing induced Fos expression in rats killed 2 h after i.c.v. injection of 1 μg of synthetic mouse Ucn II. Darkfield photomicrographs showing hybridization histochemical localization of CRF-R2 mRNA in regions corresponding to those illustrated in C and E are provided in D and F, respectively. Central Ucn injection provoked Fos induction primarily in a set of interconnected structures involved in central autonomic and neuroendocrine control, including the parvocellular division of the paraventricular nucleus (A), the central nucleus of the amygdala (B), and the nucleus of the solitary tract (NTS, C). Among these, only the NTS is a site of CRF-R2 expression (D). Other principal sites of CRF-R2 expression, including the ventromedial nucleus of the hypothalamus (F), failed to show Ucn II-induced Fos expression over the range of peptide doses examined (1–10 μg). (Magnification for all photomicrographs: ×75.)

Figure 4

Effects of central Ucn II on food intake and gross motor activity. (A) Mean (± SEM; n = 3–6 per group) cumulative nighttime food intake (g) following i.c.v. administration of 1 μg of CRF, Ucn, or Ucn II. Both CRF and Ucn significantly reduced food intake compared with saline-injected controls, beginning at 4 h postinjection, whereas the effect of Ucn II was not manifest until 6 h after treatment. *, P < 0.002 (CRF and Ucn vs. saline); **, P < 0.002 (CRF, Ucn, and Ucn II vs. saline). (B) Telemetric measures of gross motor activity were significantly elevated in animals that received i.c.v. injections of CRF; neither Ucn nor Ucn II significantly affected motor activity. *, P < 0.001 (CRF vs. saline).