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Corticotropin-releasing factor (CRF) mediates many critical aspects of the physiological response to stress. These effects are elicited by binding to specific highaffinity receptors, which are coupled to guanine nucleotide stimulatory factor (Gs)-response pathways. Recently, a gene encoding a receptor for CRF, expressed in pituitary and the central nervous system (PC-CRF receptor), was isolated and characterized. Here we report the identification and characterization of a second, distinct CRF receptor that is expressed primarily in heart and skeletal muscle and exhibits a specific ligand preference and antagonist sensitivity compared with the PC-CRF receptor. We refer to this second receptor as the heart/muscle (HM)-CRF receptor.Corticotropin-releasing factor (CRF) (1) is a member of a family of peptides from different species that act as agonists of the CRF receptor. These peptides include the frog skin peptide, sauvagine (2), and the teleost fish urophysis peptide, urotensin I (3). CRF is a 41-amino acid hypothalamic neuropeptide that plays a central role in coordinating the communications between endocrine, nervous, and immune systems to achieve homeostasis in response to environmental adversities (4, 5). The peptide was originally characterized in the hypothalamo-hypophyseal system but was later found to be widely distributed throughout the central nervous system (CNS), where it appeared to function as a neurotransmitter or neuromodulator (6). In the CNS, CRF initiates the hypothalamic-pituitary-adrenal axis by stimulating the release of adrenocorticotropin (ACTH) and ,B-endorphin from the anterior pituitary. ACTH stimulates adrenal cortex to secrete corticosteroids that, in turn, elicit a wide range of biological responses and exert negative feedback on the hypothalamus and pituitary (4, 5, 7). Both sauvagine and urotensin I have been shown to stimulate the hypothalamic-pituitary-adrenal axis after i.v. administration (8). Intracerebroventricular administration of CRF provokes stress-like responses including activation of the sympathetic nervous system, resulting in an elevation of plasma epinephrine, norepinephrine, and glucose, which results in increased heart rate and mean arterial blood pressure (9, 10). Outside the CNS, CRF immunoreactivity is detectable in multiple peripheral organs, including placenta, adrenal medulla, pancreas, lung, stomach, duodenum, and liver (4, 5). i.v. administration of CRF, sauvagine, and urotensin I has been shown to elicit peripheral systemic responses, including vasodilation (11).CRF functions by binding to a membrane-bound receptor that is coupled to the guanine nucleotide stimulatory factor (G,) signaling protein, resulting in increased intracellular cAMP levels (12,13 (23), and growth hormonereleasing factor ]. The PC-CRF receptor mRNA was found to be most highly expressed in the cerebellum and pituitary and found at lower levels in other brain areas, intestine, and testes. This receptor is undetectable in other tissues examined, including heart and skeletal mus...

A pituitary LIM homeodomain factor, PLim, is expressed as Rathke's pouch forms and as specific pituitary cell phenotypes are established, suggesting functional roles throughout pituitary development. While selectively expressed in Pituitary organ commitment appears to occur shortly after a region of the somatic ectoderm makes direct contact with neuroectodermal cells in an area of mesenchymal incompetence (1, 2). Subsequently, five distinct cell types, each characterized by the expression of a unique hormone, appear in a spatially and temporally specific fashion (reviewed in refs. 3 and 4). The pituitary-specific POU-domain transcription factor Pit-1 (3, 4) is selectively activated in the caudomedial part of the nascent gland at embryonic day 15.5 (e15.5) in the mouse and is required for activation of the prolactin (Prl), growth hormone (GH), and thyroid-stimulating hormone }3-subunit (TSHI3) genes in this region, as well as for somatotrope, lactotrope, and thyrotrope cell proliferation (5-7). Additional activating factors work with Pit-1 to achieve cellspecific gene activation (8)(9)(10)(11)(12)(13)(14)(15).A second family of homeodomain transcription factors, initially defined by RNA. These RT-PCR-generated DNA fragments were used to screen a mouse pituitary cDNA library and a mouse genomic library. RNase protection assays were performed with an intron 3-containing cDNA clone (no. 11) as template for T7RNA polymerase-directed synthesis of a radiolabeled antisense probe. In situ hybridization of 20-,um sagittal sections of mouse embryos and organs (e9, e9.5, elO.5, and e15.5) fixed in buffered 10% formalin was performed as described (1) by using T7 RNA polymerase to generate two separate 35S-labeled cRNA probes (486 and 550 nt) corresponding to fragments of N-terminal or C-terminal coding sequences of P-Lim.In Vitro Protein-Protein Interaction, DNA Binding, and Transfection Assays. Restriction fragments of P-Lim cDNA were ligated in frame into pGEX-KG (39) to yield glutathione S-transferase (GST) fusion proteins, and PCR was used to generate deletions of each LIM region separately (ALIM-1, A1-87; ALIM-2, A1-29 and A86-154) and of the entire LIM region (P-ALim = A1-151).[35S]Methionine-labeled in vitro translated protein was incubated for 20 min at 37°C with 2-3 jig of GST fusion protein bound to 25 p,l of glutathioneagarose beads in a total volume of 100 ,ul of 20 mM Hepes, pH 7.9/100 mM NaCl/1 mM EDTA/4 mM MgCl2/1 mM dithiothreitol, 0.02% (vol/vol) Nonidet P-40/10% (vol/vol) glycerol/0.5% (wt/vol) nonfat dry milk, with ethidium bromide at 50 ,ug/ml to eliminate potential protein-DNA interactions (32).Protein-mediated gel shift assays, in vitro culture, and transient transfection of cells were performed as described (9,14). For the mouse a-glycoprotein subunit (aGSU) reporter gene, a BamHI-Pst I fragment containing the promoter region of the aGSU starting from bp -440 was ligated to the luciferase gene in the pGL2 basic vector (Promega). Mouse TSH,B (from kb -1.2), rat Prl promoter/enhancer, and mous...

Left-right asymmetry is initiated during chick embryogenesis in small domains near Hensen's node. Subsequently, broad asymmetric gene expression domains are established in the lateral plate mesoderm, ultimately determining the directionality of morphogenetic events. The transfer of asymmetric information from the node to the lateral plate is mediated by Caronte (Car), a novel member of the Cerberus/Dan gene family, which induces targets by antagonizing symmetrically expressed BMP signals. In addition, BMP antagonism by Car induces asymmetric expression of Lefty in the midline, preventing spread of left-sided signals to the contralateral side.