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

R Scott Struthers,Richard A Maki,Stephen J Perry,Sam R.J Hoare,Dimitri E Grigoriadis,Trudy A Kohout,Sachiko Junger

doi:10.1074/jbc.m412914200

R Scott Struthers, Richard A Maki + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m412914200

Copy DOI

Abstract

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 beta-arrestin2, and to be internalized rapidly, likely through clathrin-coated pits. Astressin, however, fails to induce receptor phosphorylation or beta-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.

Highlights

The 41-amino acid neuropeptide corticotropin releasing factor (CRF)1 is the principal regulator of the hypothalamic-pituitary-adrenal axis, and as such plays a critical role in mediat
Further modifications made to CRF truncated in this manner have produced a number of different antagonist peptides including astressin (cyclo(30 –33)-[DPhe12,Nle21,38,Glu30,Lys33]CRF-(12– 41)), a high affinity antagonist for CRF1 receptors that possesses enhanced biological stability, allowing its extensive use in vivo to dissect the functions of the CRF system [11, 12]
It has recently been reported that the class B PTH1 receptor undergoes internalization following binding to the truncated antagonist peptide parathyroid hormone (PTH)-(7–34), a process that is independent of receptor activation [23]

Summary

EXPERIMENTAL PROCEDURES

Materials—Peptides were synthesized by solid-phase methodology on a Beckman Coulter 990 peptide synthesizer (Fulton, CA) and purified as previously described [13]. The following day the cells were subjected to the appropriate drug treatments, washed twice with ice-cold internalization medium (Dulbecco’s modified Eagle’s medium containing 25 mM HEPES, pH 7.4, 0.2 mM glutamine, 1 mM sodium pyruvate, 50 IU/ml penicillin, and 50 ␮g/ml streptomycin) and incubated for 1 h at 4 °C with anti-FLAG M2 antibody (Sigma) diluted 1:500 in internalization medium. Each well was washed 4 times with ELISA wash buffer (0.05% (v/v) Tween 20 in PBS) and incubated for 2 h at room temperature with the previously described anti-CRF1 receptor antiserum 4467a-CRF1 [31] diluted 1:10,000 in antibody dilution buffer (PBS containing 1% (w/v) bovine serum albumin). The optical density of each well was read at 450 nm using an EMax microplate reader (Molecular Devices, Sunnyvale, CA)

RESULTS

CRF receptors by astressin analogs

DISCUSSION