Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG)
Corticotropin-releasing factor (CRF), 2 the main regulator of the hypothalamic pituitary adrenal axis (1), binds to specific G protein-coupled receptors CRFR1 (2, 3) and CRFR2 (4 -7) cloned from several mammalian and nonmammalian species. A third CRF receptor, CRFR3, was found only in fish (8). Several CRF-like peptides were shown to interact with the CRF receptors with high affinity: sauvagine (SVG) characterized from the frog skin (9), urotensin I from the urophysis of fish (10), and urocortin I (UCN1) from the mammalian brain (11). Later, UCN2 and UCN3 (or stresscopins) (12-14) where characterized as CRFR2-specific ligands with little or no affinity to CRFR1.The CRF receptors, which belong to group B of the G protein-coupled receptor superfamily, have six conserved cysteine residues and several N-linked glycosylations in their N-terminal extracellular domains. The extracellular cysteine residues form disulfide bridges that stabilize the structure of the N terminus (15, 16). The N-linked glycosylation moieties are important for processing through the Golgi system and cell surface expression (17). Activation of the cloned CRF receptors stimulates adenylate cyclase (4 -7), phospholipase C (18), and mitogen-activated protein kinases (MAPKs) (19,20).Chemical and/or photoaffinity cross-linking have been extensively used for characterization of ligand-receptor interaction within group B of the G protein-coupled receptor superfamily (21-25), including the CRF receptors (26,27). Recently, the photosensitive amino acid analog p-benzoylphenylalanine (Bpa) was used to substitute several residues within the UCN1 sequence, and photoaffinity cross-linking was used to map the interaction site; the data showed that the very N-terminal ligand residues (1-11) that are responsible for receptor activation are in a close proximity to the second extracellular loop within the juxtamembrane (J-) domain of CRFR1 (26). These data contradict the proposed model derived from the interaction of the free receptor N terminus with the ligand (28 -31). We have previously used an oxidation-resistant SVG analog, [Tyr 0 , Gln 1 , Leu 17 ]SVG (YQL-SVG), which binds to CRFR1 and CRFR2 with high affinity and which cross-links to the CRF receptors with a high efficiency (32), to map the disuccinimidyl suberate-cross-linked residues, and identified covalent crosslinking between Lys 16 of SVG and Lys 257 of CRFR1 in the second extracellular loop (27). In this paper we have used a Bpasubstituted SVG analog, [Tyr 0 , Gln 1 , Bpa 17 ]SVG (YQB-SVG), which has high affinity for CRFR1, and mapped the Bpa 17 crosslinking site to His 117 of CRFR1 and thus established that position 17 of the radioligand lies within 9 Å of position 117 located in the first transmembrane-spanning domain (TM1) of CRFR1.
Uterine leiomyomata (UL), the most common neoplasm in reproductive-age women, have recurrent cytogenetic abnormalities including del(7)(q22q32). To develop a molecular signature, matched del(7q) and non-del(7q) tumors identified by FISH or karyotyping from 11 women were profiled with expression arrays. Our analysis using paired t-tests demonstrates this matched design is critical to eliminate confounding effects of genotype and environment that underlie patient variation. A gene list ordered by genome-wide significance showed enrichment for the 7q22 target region. Modification of the gene list by weighting each sample for percent of del(7q) cells to account for the mosaic nature of these tumors further enhanced the frequency of 7q22 genes. Pathway analysis revealed two of the 19 significant functional networks were associated with development and the most represented pathway was protein ubiquitination, which can influence tumor development by stabilizing oncoproteins and destabilizing tumor suppressor proteins. Array CGH (aCGH) studies determined the only consistent genomic imbalance was deletion of 9.5 megabases from 7q22-7q31.1. Combining the aCGH data with the del(7q) UL mosacism-weighted expression analysis resulted in a list of genes that are commonly deleted and whose copy number is correlated with significantly decreased expression. These genes include the proliferation inhibitor HPB1, the loss of expression of which has been associated with invasive breast cancer, as well as the mitosis integrity-maintenance tumor suppressor RINT1. This study provides a molecular signature of the del(7q) UL subgroup and will serve as a platform for future studies of tumor pathogenesis.
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