Many hormones act on neuroendocrine cells by activating second messenger pathways. Two of these, the phosphoinositol and cAMP-dependent pathways, cause changes in cellular activity through specific protein kinases. By phosphorylating cytoplasmic and nuclear proteins, these kinases apparently coordinate cellular processes, including the biosynthesis and release of neuropeptides. Somatostatin biosynthesis and release, for example, are both positively regulated by the second messenger cAMP in hypothalamic cells, and cAMP also induces somatostatin gene transcription 8-10-fold in transfected PC12 pheochromocytoma cells. Transcriptional induction requires a 30-nucleotide cAMP response element (CRE) which is conserved in other cAMP-responsive genes. This element also confers cAMP responsiveness when placed upstream of the heterologous simian virus 40 (SV40) promoter. The somatostatin gene does not, however, respond to cAMP in mutant PC12 cells which lack cAMP-dependent protein kinase type II activity. Activation of somatostatin gene transcription may consequently require the phosphorylation of a nuclear protein which binds to the CRE. Using a DNase I protection assay, we have characterized a nuclear protein in PC12 cells which binds selectively to the CRE in the somatostatin gene. We have purified this protein which is of relative molecular mass 43,000 (Mr 43K) by sequence-specific DNA affinity chromatography. This 43K CRE binding protein (CREB) is phosphorylated in vitro when it is incubated with the catalytic subunit of cAMP-dependent protein kinase. Stimulating PC12 cells with forskolin, an activator of adenyl cyclase, causes a 3-4-fold increase in the phosphorylation of this protein. We conclude that the cAMP-dependent pathway may regulate gene transcription in response to hormonal stimulation by phosphorylating this CREB protein.

The corticotropin-releasing factor (CRF) family of neuropeptides includes the mammalian peptides CRF, urocortin, and urocortin II, as well as piscine urotensin I and frog sauvagine. The mammalian peptides signal through two G protein-coupled receptor types to modulate endocrine, autonomic, and behavioral responses to stress, as well as a range of peripheral (cardiovascular, gastrointestinal, and immune) activities. The three previously known ligands are differentially distributed anatomically and have distinct specificities for the two major receptor types. Here we describe the characterization of an additional CRF-related peptide, urocortin III, in the human and mouse. In searching the public human genome databases we found a partial expressed sequence tagged (EST) clone with significant sequence identity to mammalian and fish urocortin-related peptides. By using primers based on the human EST sequence, a full-length human clone was isolated from genomic DNA that encodes a protein that includes a predicted putative 38-aa peptide structurally related to other known family members. With a human probe, we then cloned the mouse ortholog from a genomic library. Human and mouse urocortin III share 90% identity in the 38-aa putative mature peptide. In the peptide coding region, both human and mouse urocortin III are 76% identical to pufferfish urocortin-related peptide and more distantly related to urocortin II, CRF, and urocortin from other mammalian species. Mouse urocortin III mRNA expression is found in areas of the brain including the hypothalamus, amygdala, and brainstem, but is not evident in the cerebellum, pituitary, or cerebral cortex; it is also expressed peripherally in small intestine and skin. Urocortin III is selective for type 2 CRF receptors and thus represents another potential endogenous ligand for these receptors.

Corticotropin releasing factor (CRF) is a major integrator of adaptive responses to stress. Two biochemically and pharmacologically distinct CRF receptor subtypes (CRFR1 and CRFR2) have been described. We have generated mice null for the CRFR1 gene to elucidate the specific developmental and physiological roles of CRF receptor mediated pathways. Behavioral analyses revealed that mice lacking CRFR1 displayed markedly reduced anxiety. Mutant mice also failed to exhibit the characteristic hormonal response to stress due to a disruption of the hypothalamic-pituitary-adrenal (HPA) axis. Homozygous mutant mice derived from crossing heterozygotes displayed low plasma corticosterone concentrations resulting from a marked agenesis of the zona fasciculata region of the adrenal gland. The offspring from homozygote crosses died within 48 hr after birth due to a pronounced lung dysplasia. The adrenal agenesis in mutant animals was attributed to insufficient adrenocorticotropic hormone (ACTH) production during the neonatal period and was rescued by ACTH replacement. These results suggest that CRFR1 plays an important role both in the development of a functional HPA axis and in mediating behavioral changes associated with anxiety.

Activins and inhibins, structurally related members of the TGF-beta superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7-9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-beta receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand-receptor sensitivity, selectivity and function.

Corticotropin-releasing factor (CRF; corticoliberin) regulates the secretion of corticotropin (ACTH) and 18-endorphin and has a broad range of effects on the nervous, endocrine, reproductive, cardiovascular, gastrointestinal, and immune systems. Recently, human, rat, and mouse CRF receptors (CRF-R) have been cloned and functionally and anatomically characterized. We report here the cloning of a second CRF-R cDNA (CRF-RB), which encodes a protein of431 amino acids, which is 16 amino acids longer and 68% similar to the previously cloned CRF-R, CRF-RA. When transiently expressed in COS-M6 cells, CRF-RB binds CRF with high affinity [Kd = 1.2 (0.57-2.5) nM] and transduces the CRI-stimulated signal of the accumulation of intracellular cAMP, which is inhibited by a CRF antagonist. Comparison of the amino acid sequences of CRF-RB and the previously cloned receptor reveals major differences in the N-terminal domain and in the extracellular loops, whereas the sequences of the intracellular loops are nearly identical. CRF-RB and related transcripts are expressed in the heart, as well as in other tissues, including the gastrointestinal tract, epididymis, and brain.Corticotrophin-releasing-factor (CRF; corticoliberin), the 41-amino acid peptide originally isolated from the hypothalamus (1) as the major regulator of corticotropin (ACTH) and f3-endorphin secretion by the anterior pituitary, has been shown to be widely distributed in, and to have multiple effects on, a wide variety of tissues (2, 3). Consistent with the broad range of roles proposed for CRF, high-affinity binding sites have been found in pituitary (4), brain (5, 6), adrenals (7), spleen (8), and monocytes (9). Recently, our group (10, 11) and others (12, 13) reported the cloning of CRF receptors (CRF-R), which we now refer to as CRF-RA, from pituitary and brain. These receptors belong to the seven transmembrane domain (TMD) calcitonin/vasoactive intestinal peptide/ growth hormone-releasing hormone receptor family. The distribution (14) and functionality of CRF-RA indicated that it satisfied many criteria for a physiologic CRF receptor. In a human Cushing disease tumor cDNA library, we also observed the presence of a splice variant, CRF-RA2 (10), in which 29 amino acids are inserted into the first intracellular loop.During the course of the characterization of the mouse gene encoding the CRF-R, we obtained evidence for a related gene, CRF-RB, which we partially sequenced. RNase protection analysis indicated high expression of this gene in the heart. We report here the cloning and characterization of a cDNA from a mouse heart cDNA library encoding a second CRF-R. § MATERIALS AND METHODS [a-32P]dCTP and the following primers: sense strand, 5'-CTGCATCACCACCATCTTCAACT-3'; and antisense strand, 5'-AGCCACTTGCGCAGGTGCTC-3'. The template used in generating the probe was plasmid DNA corresponding to one exon of CRF-RB extending from amino acid 206 to 246 (see Fig. 1). PCR amplification was carried out for 30 cycles (denaturation at 94°C for 1 min, annealing at 5...