Distinct Conformations of the Corticotropin Releasing Factor Type 1 Receptor Adopted following Agonist and Antagonist Binding Are Differentially Regulated

• Published in: The Journal of biological chemistry Vol. 280; no. 12; pp. 11560 - 11568
• Main Authors: Perry, Stephen J., Junger, Sachiko, Kohout, Trudy A., Hoare, Sam R.J., Struthers, R. Scott, Grigoriadis, Dimitri E., Maki, Richard A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 25.03.2005; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; CHO Cells; Corticotropin-Releasing Hormone - metabolism; Corticotropin-Releasing Hormone - pharmacology; Cricetinae; Endocytosis; Humans; Mice; Peptide Fragments - metabolism; Peptide Fragments - pharmacology; Protein Conformation; Receptors, Corticotropin-Releasing Hormone - antagonists & inhibitors; Receptors, Corticotropin-Releasing Hormone - chemistry; Receptors, Corticotropin-Releasing Hormone - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M412914200

The corticotropin releasing factor (CRF) type 1 receptor (CRF1) is a class B family G protein-coupled receptor that regulates the hypothalamic-pituitary-adrenal stress axis. Astressin is an amino-terminal truncated analog of CRF that retains high affinity binding to the extracellular domain of the receptor and is believed to act as a neutral competitive antagonist of receptor activation. Here we show that despite being unable to activate the CRF1 receptor, astressin binding results in the internalization of the receptor. Furthermore, entirely different pathways of internalization of CRF1 receptors are utilized following CRF and astressin binding. CRF causes the receptor to be phosphorylated, recruit β-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or β-arrestin2 recruitment, and internalization is slow and occurs through a pathway that is insensitive to inhibitors of clathrin-coated pits and caveolae. The fate of the internalized receptors also differs because only CRF-induced internalization results in receptor down-regulation. Furthermore, we present evidence that for astressin to induce internalization it must interact with both the extracellular amino terminus and the juxtamembrane domain of the receptor. Astressin binds with 6-fold higher affinity to full-length CRF1 receptors than to a chimeric protein containing only the extracellular domain attached to the transmembrane domain of the activin IIB receptor, yet two 12-residue analogs of astressin have similar affinities for both proteins but are unable to induce receptor internalization. These data demonstrate that agonists and antagonists for CRF1 receptors promote distinct conformations, which are then differentially regulated.