PubMed · 11582791
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K Nitta. 2001. Leczyme.. https://doi.org/10.1016/s0076-6879(01)41164-5
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Contact sites between the corticotropin-releasing factor receptor type 1 (CRFR1), the sauvagine (SVG) radioligands [Tyr(0),Gln(1)]SVG ((125)I-YQS) and [Tyr(0),Gln(1), Leu(17)]SVG ((125)I-YQLS) were examined. (125)I-YQLS or (125)I-YQS was cross-linked to CRFR1 using the chemical cross-linker, disuccinimidyl suberate (DSS), which cross-links the epsilon amino groups of lysine residues that have a molecular distance of 11.4 A. DSS specifically and efficiently cross-linked (125)I-YQLS and (125)I-YQS to CRFR1. CRFR1 contains 5 putative extracellular lysine residues (Lys(110), Lys(111), Lys(113), Lys(257), and Lys(262)) that can cross-link to the 4 lysine residues (Lys(16), Lys(22), Lys(25), and Lys(27)) of the radioligands. Identification of the CNBr-cleaved fragments of CRFR1 cross-linked to (125)I-YQLS or (125)I-YQS established that the second extracellular loop of CRFR1 cross-links to Lys(16) of YQS. Additionally, site-directed mutagenesis (changing Lys to Arg in CRFR1 individually and in combination) revealed that Lys(257) in the second extracellular loop of CRFR1 is an important cross-linking site. In conclusion, it was shown that in SVG-bound CRFR1, Lys(257) of CRFR1 lies in close proximity (11.4 A) to Lys(16) of SVG.
The wood frog Rana sylvatica survives for weeks during winter hibernation with up to 65% body water frozen as ice. Natural freeze tolerance includes both seasonal and freeze-induced molecular adaptations that control ice formation, deal with long-term ischemia, regulate cell volume changes, and protect macromolecules. This report identifies and characterizes a novel freeze-inducible gene, li16, that codes for a protein of 115 amino acids. Northern blot analysis showed that li16 transcript levels rose quickly during freezing to reach levels 3.7-fold higher than control values after 24 h; immunoblotting showed a parallel 2.4-fold rise in Li16 protein. Regulatory influences on gene expression were assessed. Nuclear runoff assays confirmed that freezing initiated an increase in the rate of li16 transcription, and analysis of signal transduction pathways via in vitro incubation of liver slices implicated a cGMP-mediated pathway in li16 expression. Gene and protein expression in liver was also strongly stimulated by anoxia exposure, whereas the gene was less responsive to dehydration stress. The strong response of li16 to both freezing and anoxia, and the rapid down-regulation of the gene when oxygen was reintroduced, suggest that the Li16 protein may play a role in ischemia resistance during freezing.
Although corticotropin-releasing hormone (CRH), a regulator of stress responses, acts through two receptors (CRH1 and CRH2), the role of CRH2 in stress responses remains unclear. Knock-out mice without the CRH2 gene exhibit increased stress-like behaviors. This profile could result either directly from the absence of CRH2 receptors or indirectly from developmental adaptations. In the present study, CRH2 receptors were acutely blocked by alpha-helical CRH (alpha(h)CRH, CRH1/CRH2 antagonist; 0, 30, 100, and 300 ng) infusion into the lateral septum (LS), which abundantly expresses CRH2 but not CRH1 receptors. Freezing, locomotor activity, and analgesia were tested after infusion. Intra-LS alpha(h)CRH blocked shock-induced freezing without affecting activity or pain responses; infusions into lateral ventricle or nucleus of the diagonal band had no effects. The same behavioral profile was obtained with d-Phe-CRH((12-41)) (100 ng), another CRH1/CRH2 antagonist. A selective CRH1 antagonist (NBI27914), in doses that reduced freezing on intra-amygdala (central nucleus) infusion (0, 0.2, and 1.0 microg), did not affect freezing when infused into the LS. Ex vivo autoradiography revealed that binding of [125I]sauvagine, a mixed CRH1/CRH2 agonist, was prevented in the LS by previous intra-LS infusion of alpha(h)CRH but not NBI27914. In vitro studies demonstrated that [125I]sauvagine binding in the LS could be inhibited by a CRH1/CRH2 antagonist but not by the selective CRH1 receptor antagonist, confirming that in the LS, alpha(h)CRH antagonized exclusively CRH2 receptors. Acute antagonism of CRH2 receptors in the LS thus produces a behaviorally, anatomically, and pharmacologically specific reduction in stress-induced behavior, in contrast to results of recent knock-out studies, which induced congenital and permanent CRH2 removal. CRH2 receptors may thus represent a potential target for the development of novel CRH system anxiolytics.