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Expression, binding, and signaling properties of CRF 2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage‐dependent regulation of functional receptors
Author(s) -
Gutknecht Eric,
Hauger Richard L.,
Van der Linden Ilse,
Vauquelin Georges,
Dautzenberg Frank M.
Publication year - 2008
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.2007.05052.x
Subject(s) - receptor , urocortin , agonist , antagonist , stimulation , endocrinology , medicine , receptor antagonist , biology , endogeny , ic50 , retinoblastoma , microbiology and biotechnology , chemistry , in vitro , biochemistry , gene
Endogenous expression of the corticotropin‐releasing factor type 2a receptor [CRF 2(a) ] but not CRF 2(b) and CRF 2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF 2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF 1 receptor was expressed at higher levels than the CRF 2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF 1 ‐preferring or non‐selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of ∼100 pmoles/10 5 cells, whereas the exclusive CRF 2 receptor‐selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of ∼25–30 pmoles/10 5 cells. UCN2 and 3‐mediated cAMP stimulation was potently blocked by the ∼300‐fold selective CRF 2 antagonist antisauvagine (IC 50 = 6.5 ± 1.6 nmol/L), whereas the CRF 1 ‐selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 μmol/L. When the CRF 1 ‐preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC 50 = 38.4 ± 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC 50 = 2.04 ± 0.2 μmol/L). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF 2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF 1 and CRF 2(a) receptors and that the CRF 2(a) receptor protein is up‐regulated during prolonged culture.