Soluble Corticotropin-Releasing Hormone Receptor 2α Splice Variant Is Efficiently Translated But Not Trafficked for Secretion

Abstract

CRH directs the physiological and behavioral responses to stress. Its activity is mediated by CRH receptors (CRH-R) 1 and 2 and modulated by the CRH-binding protein. Aberrant regulation of this system has been associated with anxiety disorders and major depression, demonstrating the importance of understanding the regulation of CRH activity. An mRNA splice variant of CRH-R2alpha (sCRH-R2alpha) was recently identified that encodes the receptor's ligand-binding extracellular domain but terminates before the transmembrane domains. It was therefore predicted to serve as a secreted decoy receptor, mimicking the ability of CRH-binding protein to sequester free CRH. Although the splice variant contains a premature termination codon, predicting its degradation by nonsense-mediated RNA decay, cycloheximide experiments and polysome profiles demonstrated that sCRH-R2alpha mRNA escaped this regulation and was efficiently translated. However, the resulting protein was unable to serve as a decoy receptor because it failed to traffic for secretion because of an ineffective signal peptide and was ultimately subjected to proteosomal degradation. Several other truncated splice variants of G protein-coupled transmembrane receptors regulate the amount of full-length receptor expression through dimerization and misrouting; however, receptor binding assays and immunofluorescence of cells cotransfected with sCRH-R2alpha and CRH-R2alpha or CRH-R1 indicated that sCRH-R2alpha protein does not alter trafficking or binding of full-length CRH-R. Although sCRH-R2alpha protein does not appear to function as an intracellular or extracellular decoy receptor, the regulated unproductive splicing of CRH-R2alpha pre-mRNA to sCRH-R2alpha may selectively alter the cellular levels of full-length CRH-R2alpha mRNA and hence functional CRH-R2alpha receptor levels.

The Pseudo Signal Peptide of the Corticotropin-releasing Factor Receptor Type 2A Prevents Receptor Oligomerization

“…The CRF 2(a) R instead possesses a pseudo signal peptide, which is unable to mediate ER targeting, remains uncleaved, and forms an additional hydrophobic domain at the N tail of the receptor, which is so far unique within the GPCR protein family (11)(12)(13)(14). Conventional signal peptide functions are blocked by a single amino acid residue (Asn 13 ), and mutation of this residue causes conversion to a cleaved signal peptide (11).…”

Topographical distribution of corticotropin-releasing factor type 2 receptor-like immunoreactivity in the rat dorsal raphe nucleus: co-localization with tryptophan hydroxylase

“…The antibody that was used is likely to recognize all known isoforms of the CRF 2 receptor because the antibody recognizes a 12-amino acid sequence of the N-terminus that is nearly identical in all CRF 2 receptor isoforms that have been characterized (Figure 1). A recent study (Evans and Seasholtz, 2009) has shown that the sCRF 2α receptor is found in the rat brain, and that the mRNA is efficiently translated. In addition Evans and Seasholtz (2009) showed that the sCRF 2α receptor is not trafficked to the membrane, which may account in part for the apparent abundance of cytoplasmic staining seen in this study and previous studies (Waselus et al, 2009).…”

“…A recent study (Evans and Seasholtz, 2009) has shown that the sCRF 2α receptor is found in the rat brain, and that the mRNA is efficiently translated. In addition Evans and Seasholtz (2009) showed that the sCRF 2α receptor is not trafficked to the membrane, which may account in part for the apparent abundance of cytoplasmic staining seen in this study and previous studies (Waselus et al, 2009). Consistent with these findings, recent studies by Schulz and colleagues (2010) showed that CRF 2α receptor contains an N-terminal pseudo signal peptide that is unable to target the peptide to the endoplasmic reticulum membrane, resulting in very low cell surface expression (Schulz et al, 2010).…”

“…: EDL88098), mCRF 2α (Chen et al, 2005a) (NCBI Protein database accession no. : AAS07021), mCRF 2αsol (Chen et al, 2005b; Evans and Seasholtz, 2009), (NCBI Protein database accession no. : AAU94301), rCRF 2α -tr receptor (Miyata et al, 1999), rCRF 2α (Lovenberg et al, 1995b) (NCBI Protein database accession no.…”

“…These isoforms have distinct expression patterns and binding affinities for CRF and its family of peptides (Lovenberg et al, 1995a; Kostich et al, 1998; Miyata et al, 1999; Miyata et al, 2001; Chen et al, 2005b; Evans and Seasholtz, 2009). …”