The Corticotropin-releasing Factor Receptor Type 2a Contains an N-terminal Pseudo Signal Peptide

Claudia Rutz,Armin Renner,Martina Alken,Katharina Schulz,Michael Beyermann,Burkhard Wiesner,Walter Rosenthal,Ralf Schülein

doi:10.1074/jbc.m601554200

Abstract

The corticotropin-releasing factor receptor type 2a (CRF(2(a)) receptor) belongs to the family of G protein-coupled receptors. The receptor possesses a putative N-terminal signal peptide that is believed to be cleaved-off after mediating the endoplasmic reticulum targeting/insertion process, like the corresponding sequence of the homologous CRF(1) receptor. Here, we have assessed the functional significance of the putative signal peptide of the CRF(2(a)) receptor and show that it is surprisingly completely incapable of mediating endoplasmic reticulum targeting, despite meeting all sequence criteria for a functional signal by prediction algorithms. Moreover, it is uncleaved and forms part of the mature receptor protein. Replacement of residue Asn(13) by hydrophobic or positively charged residues converts the sequence into a fully functional and cleaved signal peptide demonstrating that conventional signal peptide functions are inhibited by a single amino acid residue. Deletion of the domain leads to an increase in the amount of immature, intracellularly retained receptors demonstrating that the sequence has adopted a new function in receptor trafficking through the early secretory pathway. Taken together, our results identify a novel hydrophobic receptor domain in the family of the heptahelical G protein-coupled receptors and the first pseudo signal peptide of a eukaryotic membrane protein. Our data also show that the extreme N termini of the individual CRF receptor subtypes differ substantially.

Highlights

The corticotropin-releasing factor (CRF)3 receptor family is involved in the regulation of the hypothalamic-pituitary-adrenal stress axis in mammals [1,2,3]
The N tail of a membrane protein is synthesized in the cytoplasm, because translation continues until the signal anchor sequence (TM1) appears
In the case of GPCRs, it was shown that the signal peptide of the endothelin B (ETB) receptor is essential for N tail translocation across the ER membrane [10]

Summary

EXPERIMENTAL PROCEDURES

Materials—The cDNA encoding the rat CRF1 and CRF2(a) receptors were a gift from U. CRF2(a) encode GFP fusions to the N tail of the CRF2(a) receptor (position Ala121) with and without signal peptide, respectively, in the vector pSecTag2A. Plasmids CRF1.NT and p⌬SP-CRF1.NT encode the corresponding GFP fusions to the N tail of the CRF1 receptor (position Ala119) with and without signal peptide, respectively, in the vector pSecTag2A. CRF2(a).NT.PrP encodes a fusion of the N-terminal 118 amino acids of the CRF2(a) receptor to the hamster prion protein marker PrP(A120L) [15]. The plasmid p⌬SP-CRF2(a) encodes the signal peptide mutant of this receptor lacking the N-terminal 18 amino acid residues. The plasmid p⌬SP-CRF1 encodes the corresponding signal peptide mutant lacking the N-terminal 24 amino acid residues. SH-SY5Y cells were cultivated in Dulbecco’s modified Eagle’s medium/Ham’s (1:1) F-12 medium containing 10% (v/v) heat-inactivated fetal calf serum, penicillin (100 units/ml), streptomycin (100 ␮g/ml), and 1% (w/v) nonessential amino acids. Transfection of the cells with LipofectamineTM 2000 or FuGENE 6 was carried out according to the supplier’s recommendations

Confocal Laser Scanning Microscopy and Localization of GFP

RESULTS

The Pseudo Signal Peptide Does Not Influence the Ligand

Findings

DISCUSSION