Expression of the iv-mCRFR2β transcript in mouse heart. A) Representative image of electrophoretic analysis of the semiquantitative RT-PCR for mCRFR2β, iv-mCRFR2β, and the ribosomal protein S16 mRNA. B) A highly specific antiserum raised in rabbit using a synthetic peptide fragment encoding the unique C-terminal tail of iv-mCRFR2β (aa 396-420) was used for immunoblot analysis, cytoimmunostaining, and immunohistochemistry (a). Western immunoblot of iv-mCRFR2β protein isolated from transiently transfected COS-M6 cells (b). Lanes 1, 2, and 3 correspond to empty control vector, iv-mCRFR2β, and mCRFR2β constructs, respectively. C) Immunofluorescence staining of COS-M6 cells transiently transfected with iv-mCRFR2β construct and stained with anti-iv-mCRFR2β serum followed by a Cy3-conjugated secondary antibody (b). Cells transiently transfected with empty control vector (a) or mCRFR2β-myc (c) and stained with anti-iv-mCRFR2β serum followed by a Cy3-conjugated secondary antibody did not show any staining. Same mCRFR2β-myc transiently transfected cells were stained with monoclonal antimyc antibody followed by a Cy2-conjugated secondary antibody (d). D) Western immunoblot of iv-mCRFR2β protein isolated from heart (right lane) or skin (left lane) tissue extracts. E) Immunoperoxidase staining for iv-CRFR2β in mouse heart (a, c). Incubation of control sections with normal rabbit serum did not result in any staining (b, d).

Dominant-negative effects of the iv-mCRFR2β isoform. A) Activation of cAMP response element-luciferase reporter by Ucn 2 in HEK293T cells transiently cotransfected with a fixed amount (0.75 μg) of mCRFR2β construct, a luciferase reporter containing a fragment of the cAMP response element promoter of the EVX1 gene, and increasing amounts of iv-mCRFR2β construct (0.1–0.75 μg). Transfections were supplemented with empty pcDNA3 vector to control for total DNA transfected. Luciferase activity was measured 3 h after treatment with 10 nM Ucn 2. B) Activation of cAMP response element-luciferase reporter by Ucn 2 in HEK293T cells transiently cotransfected with 1:0, 1:0.5, and 1:1 ratios of mCRFR2β and iv-mCRFR2β, respectively. Luciferase reporter containing a fragment of the cAMP response element promoter was cotransfected into HEK293T cells with mCRFR2β and iv-mCRFR2β. Luciferase activity was measured 3 h after treatment with 0.001–100 nM Ucn 2. C) Surface expression of mCRFR2β measured by ELISA assay using monoclonal anti-myc antibody in HEK293T cells transiently transfected with 1:0, 1:0.5, and 1:1 ratios of mCRFR2β and iv-mCRFR2β, respectively. D) Immunofluorescence staining of COS-M6 cells transiently transfected with mCRFR2β-myc and stained with monoclonal anti-myc antibody followed by a Cy3-conjugated secondary antibody; note the full membrane expression of mCRFR2β (a). Immunofluorescence staining of COS-M6 cells transiently cotransfected with mCRFR2β-myc and iv-mCRFR2β (b–d) and double-stained with monoclonal anti-myc antibody followed by a Cy3-conjugated secondary antibody (b), and anti-iv-mCRFR2β serum followed by a Cy2-conjugated secondary antibody (c). Note the mCRFR2β retention in the ER-Golgi region and the colocalization with iv-mCRFR2β (merge and DAPI staining; d). Graphs shows mean of 8 replicates from one experiment.

Sequence and schematic structure representation of iv-mCRFR2β. A) Schematic representation of the structure of the mCRFR2 gene, including the nucleotide and translated amino acid sequences of an exon inserted in intron 13. B) Schematic representation of the structure of the known mCRFR2β functional transcript (top) and the novel iv-mCRFR2β isoform. Locations of the translation start sites (ATG) are indicated for both transcripts. Exons coding for the N-terminal extracellular domain, the seven transmembrane domains, and the C-terminal cytoplasmic domain are indicated. Hatched boxes indicate 5′- and 3′-untranslated regions; open boxes represent coding regions. C) Representative image of electrophoretic analysis of the pCRII-TOPO vector containing the known mCRFR2β transcript (clones 1, 2, and 4) and the 127-bp-longer iv-mCRFR2β transcript (clone 3), isolated from a mouse heart. Oligonucleotide primers location used for iv-mCRFR2β isolation are indicated in A (P, primer; RP, reverse primer). D) Schematic representation of the amino acid sequence of the mCRFR2β and the iv-mCRFR2β, demonstrating a unique 35- vs. 46-aa intracellular tail for iv-mCRFR2β and mCRFR2β, respectively.

Functional analysis and cellular localization of iv-mCRFR2β. A) Activation of a cAMP response element-luciferase reporter by Ucn 2 in HEK293T cells transiently transfected with mCRFR2β or iv-mCRFR2β. Luciferase reporter containing a fragment of the cAMP response element promoter of the EVX1 gene was cotransfected into HEK293T cells with mCRFR2β or iv-mCRFR2β. Luciferase activity was measured 3 h after treatment with 0.01–100 nM Ucn 2. B) Surface expression of mCRFR2β and iv-mCRFR2β measured by immunofluorescence. COS-M6 cells were transiently transfected with iv-mCRFR2β-myc (a, b) or mCRFR2β-myc (c, d) constructs, with or without Triton, and stained with monoclonal anti-myc antibody followed by a Cy3-conjugated secondary antibody. C) Surface expression of mCRFR2β and iv-mCRFR2β measured by ELISA assay using monoclonal anti-myc antibody in HEK293T cells transiently transfected with mCRFR2β-myc or iv-mCRFR2β-myc constructs. D) Cellular localization of iv-mCRFR2β. COS-M6 cells were transiently transfected with iv-mCRFR2β and double stained with anti-iv-mCRFR2β serum and either anti-p115 (a–c) or anti-PDI (d–f) as Golgi or ER markers, respectively. Graphs shows mean of 8 replicates from one experiment. ***P < 0.001.

Differential regulation of iv-mCRFR2β and mCRFR2β mRNA in mouse heart following chronic variable stress. mRNA levels of iv-mCRFR2β and mCRFR2β expressed by the mouse heart, with or without exposure to chronic variable stress (CVS) were quantified by real time PCR analysis. mCRFR2β mRNA expression levels in mouse heart were significantly down-regulated in the CVS group compared to the control group (A) while iv-mCRFR2β mRNA expression levels were significantly up-regulated (B). Data are means ± se; n = 5/group. *P < 0.05, **P < 0.01; Student’s t test.