The present study reports the isolation of a cDNA clone that encodes a second member of the corticotropin-releasing factor (CRF) receptor family, designated as the CRF2 receptor. The cDNA was identified using oligonucleotides of degenerate sequence in a PCR paradigm. A PCR fragment obtained from rat brain was utilized to isolate a full-length cDNA from a rat hypothalamus cDNA library that encoded a 411-amino acid protein with "70% identity to the known CRF1 receptor over the entire coding region. When expressed in mouse Ltk-cells, this receptor stimulates cAMP production in response to CRF and known CRF-like agonists. CRF and the nonmammalian CRF-related peptides sauvagine and urotensin I stimulate adenylate cyclase activity in a dose-dependent manner with a rank order of potency different from that of the CRF1 receptor: sauvagine > urotensinrat/human CRF > ovine CRF. Tissue distribution analysis of the mRNAs by reverse transcriptase-PCR shows CRF2 receptor mRNA is present in rat brain and detectable in lung and heart. In situ hybridization studies indicate specific expression within the brain in the ventromedial nuclei of the hypothalamus, the lateral septum, the amygdala, and entorhinal cortex, but there is unremarkable expression in the pituitary. An additional splice variant of the CRF2 receptor with a different N-terminal domain has been identified by PCR, encoding a putative protein of 431 amino acids. Thus, the data demonstrate the presence of another functional CRF receptor, with significant differences in the pharmacological profile and tissue distribution from the CRF1 receptor, which would predict important functional differences between the two receptors.Corticotropin-releasing factor (CRF), a 41-amino acid peptide, regulates the secretion of adrenocorticotropin and other proopiomelanocortin products from the anterior pituitary. CRF also coordinates the endocrine, behavioral, and autonomic responses to stress. Within the past few years, substantial evidence has accumulated from both laboratory and clinical studies implicating CRF as a physiological mediator of stress responses and stress-induced disorders (1-6). Immunocytochemical studies have shown that CRF is found within the paraventricular nucleus of the hypothalamus as well as limbic areas such as the central and medial nuclei of the amygdala, the bed nucleus of the stria terminalis, substantia inominata, septum, preoptic area, the lateral hypothalamus, and brain stem nuclei involved in stress responses and regulation of autonomic function, such as the locus coeruleus, the parabrachial nucleus, and the dorsal vagal complex (see ref. 7). CRF, when administered intracerebroventricularly, results in behavioral, physiological, and autonomic responses that are similar to those observed when animals are exposed to a stressful environment (4-6).

As a treatment for dyslipidemia, oral doses of 1-3 grams of nicotinic acid per day lower serum triglycerides, raise high density lipoprotein cholesterol, and reduce mortality from coronary heart disease ( Tavintharan

Abstract. Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system . We have established a cell culture model of the bloodbrain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP These cells form

␣ 4 ␤ 1 integrin (VLA-4) appears to be unique among the leukocyte integrins in that it can initiate the adhesion of circulating lymphocytes without cellular activation. It is not known how lymphocytes or other cell types maintain constitutive levels of ␣ 4 ␤ 1 integrin activity. The current report describes a monoclonal antibody, 15/7, that recognizes a high affinity or ligand-occupied conformation of ␤ 1 integrin. Studies with 15/7 revealed that ␣ 4 ␤ 1 integrin-dependent adhesion of leukocytic cell lines is mediated by a population of low affinity receptors that is conformationally responsive to ligand; the 15/7 epitope could be induced by nanomolar concentrations of soluble VCAM-1 or by micromolar concentrations of a peptide derived from the type III connecting segment domain of fibronectin (as ligands for ␣ 4 ␤ 1 integrin). The same receptors were also responsive to adhesion activating reagents, such as Mn 2؉ , activating anti-␤ 1 integrin antibodies, and phorbol myristate acetate, which induced the 15/7 epitope directly and/or decreased the concentration of ligand required for epitope induction. In addition to the responsive receptor pool, cells expressed a second population of ␣ 4 ␤ 1 integrin that was conformationally restrained, failing to respond to ligand or to any of the activating reagents. The relative size of the responsive and inactive receptor pools, as well as the affinity of the responsive receptors, represented a stable phenotype of different cell types and played important roles in defining the cells' adhesive capacity and ligand specificity. Similar receptor populations were measured on lymphocyte subsets in whole blood. These studies provide insight into how cells maintain different constitutive levels of ␣ 4 ␤ 1 integrin activity, and how the activity of ␤ 1 integrin can be modulated by activators of cell adhesion.Integrins are heterodimeric adhesion molecules that contribute to the specificity of cellular interactions through the recognition of numerous matrix and cell-associated ligands (1). Importantly, the ligand binding activity of integrins can be modulated rapidly, allowing cells to specify the timing and location of integrin-mediated adhesive interactions. On circulating leukocytes, for example, the ␤ 2 integrins LFA-1 and Mac-1 are thought to be activated by site-specific factors (cytokines or other adhesive interactions) during transient cell interactions with the vascular wall; the receptors then establish firm adhesive contacts and mediate leukocyte extravasation (2, 3). Likewise, platelets are stimulated at sites of vascular injury allowing ␣II b ␤ 3 integrin to bind to fibrinogen and initiate thrombosis (4).The regulation of ␣ 4 integrin activity on circulating immune cells appears to be different from that of the other leukocyte integrins described above. ␣ 4 ␤ 1 and ␣ 4 ␤ 7 integrin can mediate leukocyte adhesion to their endothelial ligands VCAM-1 (5) and MAdCAM-1 (6), respectively, without cellular activation, and can do so even in the presence of the shear forces encount...

We have attempted to ascertain the proportion of the rat hepatic genome that is under the selective influence of thyroid hormones and to describe the response patterns of individual mRNA sequences in the transition between hypothyroidism and euthyroidism and between euthyroidism and hyperthyroidism. Poly(A)+RNA was extracted from livers of thyroidectomized, intact, euthyroid rats and of thyroidectomized rats rendered euthyroid and hyperthyroid with daily doses oftriiodothyronine. The extracted RNA was translated in a reticulocyte lysate system in the presence of [3S]methionine, and the products were analyzed by two-dimensional gel electrophoresis. Triiodothyronine attenuates as well as augments the expression of certain genes at a pretranslational level. This could represent either a direct or an indirect action ofthe hormone. Triiodothyronine influences approximately 8% of the 231 mRNA sequences visualized, stimulating activity in 11 and inhibiting activity in 7 sequences. Translational activity of at least one mRNA sequence decreased in both thyroidectomized and hyperthyroid animals, compared to euthyroid levels. The relationship of mRNA response to receptor occupancy varied with examples of linear and amplified responses and responses that were maximal at less than full nuclear occupancy. (4) have also shown that T3 inhibits the relative rate of synthesis of histidinase in the rat liver. In none of these studies was the mechanism of inhibition clarified.Prompted by these observations, we undertook studies to determine whether such inhibition occurred at a posttranslational or at a pretranslational level. Both inhibitory and stimulatory effects of thyroid hormone on the genome were reflected in two-dimensional gel electrophoresis patterns of in vitro translational products of hepatic poly(A)+RNA obtained from rats ofdifferent thyroid status. These experiments allowed us to define 19 mRNA sequences under the selective influence of T3 and to document a broad spectrum of changes that occur in the transitions between the hypothyroid and euthyroid and the euthyroid and hyperthyroid states. Total cellular RNA was extracted from frozen (-800C) liver with phenol/chloroform at pH 9 as described (8). Total cellular RNA samples were resuspended in water, heated to 680C for 2 min, and applied to oligo(dT)-cellulose columns for isolation of poly(A)+RNA (9). METHODSRabbit reticulocyte lysate was prepared by the method of Evans and Lingrel (10). The lysate was treated with micrococcal nuclease (P-L Biochemicals) at 30 ,ug/ml as described by Pelham and Jackson (11). In vitro translational assays were performed as described (8). The reaction mixtures were incubated for 90 min at 230C and centrifuged at 100,000 x g for 1 hr to remove ribosomes. Incorporation of [3S]methionine into protein was determined by 10% trichloroacetic acid precipitation of an aliquot of the translated products (12). Samples of translated products containing an equal radioactivity (250,000-300,000 cpm) were then subjected to two-dimensional gel...

No abstract

Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.

Two CRF receptor subtypes (CRF1 and CRF2 receptors) with distinct brain localizations and pharmacological profiles have recently been cloned and characterized. For the CRF2 receptor subtype, at least 2 splice forms with different 5'-coding sequences (CRF2 alpha and CRF2 beta) have been identified in rat. In this article, we report the genomic structure and the corresponding complementary DNA (cDNA) sequence of the human CRF2 receptor. The gene coding for human CRF2 receptor consists of at least 12 exons and spans approximately 30 kilobases. The cDNA sequence in the protein-coding region is 94% identical to that of the reported rat CRF2 alpha receptor. At present, there is no evidence for the existence of a CRF2 beta receptor homolog in humans. The encoded receptor is 411 amino acids in length and is 70% identical to the human CRF1 receptor, with least sequence homology in the N-terminal extracellular domain (47% identical). Cells transfected with the full-length human CRF2 receptor cDNA responded to rat/human CRF and sauvagine by increasing the intracellular cAMP level, with EC50 values of approximately 20 and 1 nM, respectively. The CRF- and sauvagine-induced accumulation of intracellular cAMP could be competitively inhibited by the CRF receptor antagonist D-Phe-CRF. This pharmacological profile was comparable to that of the rat CRF2 alpha receptor. The relative abundance of the CRF2 receptor messenger RNA appears to be lower in humans than in rats for the tissues studied thus far.