CRF binding protein facilitates the presence of CRF type 2α receptor on the cell surface

Abstract

Corticotropin releasing factor binding protein (CRF-BP) was originally recognized as CRF sequestering protein. However, its differential subcellular localization in different brain nuclei suggests that CRF-BP may have additional functions. There is evidence that CRF-BP potentiates CRF and urocortin 1 actions through CRF type 2 receptors (CRF2R). CRF2R is a G protein-coupled receptor (GPCR) that is found mainly intracellularly as most GPCRs. The access of GPCRs to the cell surface is tightly regulated by escort proteins. We hypothesized that CRF-BP binds to CRF2R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF2R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF2αR in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF2R naturally colocalize in the neurites of cultured mesencephalic neurons. Heterologous expression of each protein showed that CRF-BP was localized mainly in secretory granules and CRF2αR in the endoplasmic reticulum. In contrast, CRF-BP and CRF2αR colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF2αR but not the CRF2βR isoform, increasing CRF2αR on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.

Keywords: CRH; accessory protein; escort protein; protein interactions.

Fig. 1.

CRF-BP and CRF₂R colocalize in cultured mesencephalic neurons. (A) Confocal immunodetection for CRF-BP, CRF₂R, and the neuronal marker β-III-tubulin (β-III-tub). Arrows depict stained nonneuronal cells. (Scale bar, 5 µm.) (B) Confocal images for CRF-BP, CRF₂R, and merge showing colocalization in soma (a₁–c₁) and neurites (a₂–c₂). (Scale bar, 5 µm for soma and 8 µm for neurites.) (C and D) Van Steensel´s analyses (C) and Pearson’s coefficient (D) of CRF-BP and CRF₂R colocalization. The soma and two to three neurites from 10 neurons of three independent experiments were analyzed. Data indicate the mean ± SEM (*P < 0.05).

Fig. 2.

CRF-BP interacts with CRF_2αR. (A) Coimmunoprecipitation of ^FlagCRF-BP and ^MycCRF_2αR (Myc-_2αR) or ^HACRF_2βR (HA-_2βR) from HEK293T cell lysates. BP, CRF-BP; R2, CRF₂R. (B) Yeast two-hybrid assay of CRF-BP and N-terminal domain of CRF_2αR (Nter-_2αR).

Fig. 3.

CRF-BP is localized mainly in secretory granules and CRF_2αR in the endoplasmic reticulum. (A and C) Van Steensel´s colocalization analysis of CRF-BP^Flag (A) and ^HACRF_2αR (C) with the subcellular markers: calnexin, clathrin, giantin, SgII, and TfR. Data were obtained from 30 cells of three independent experiments. (B and D) Confocal immunodetection for CRF-BP plus SgII (B) or CRF_2αR plus calnexin (D). Magnifications of the region in the Insets are shown. (Scale bar, 5 µm.)

Fig. 4.

CRF_2αR retained CRF-BP in the endoplasmic reticulum when coexpressed. (A and C) Confocal immunodetection for CRF-BP and SgII (A) or calnexin (C). Magnifications of the region in the Insets are shown. (Scale bar, 5 µm.) (B and D) Van Steensel´s colocalization analysis of CRF-BP with SgII (B) or calnexin (D). Data were obtained from 10 cells of three independent experiments.

Fig. 5.

CRF-BP is retained in the ER when coexpressed with CRF_2αR but not with CRF_2βR. (A and B) Confocal immunodetection for CRF-BP and HA epitope. The detection of ^HACRF_2αR is shown in A and ^HACRF_2βR in B. Magnifications of the region in the Insets are shown. (Scale bar, 5 µm.) (C) Van Steensel's colocalization analysis of CRF-BP^Flag with ^HACRF_2αR or ^HACRF_2βR. Data were obtained from 10 cells of four independent experiments. (D) Confocal immunodetection for CRF-BP and calnexin in cells coexpressing CRF-BP^flag and ^HACRF_2βR. Magnification of the region in the Inset is shown. (Scale bar, 5 µm.) (E) Pearson's correlation coefficient of CRF-BP with calnexin obtained from images of PC12 cells transfected with CRF-BP^flag alone (pcDNA) or cotransfected with ^HACRF_2αR or ^HACRF_2βR. Data were obtained from 10 cells of three independent experiments. Data indicate the mean ± SEM (**P < 0.01).

Fig. 6.

CRF-BP increases CRF_2αR in the plasma membrane. (A) Confocal immunodetection for HA and biotin in nonpermeabilized cells. (Scale bar, 5 µm.) (B and C) Confocal immunodetection for membrane and total HA and quantification of the ^HACRF₂R membrane/total fluorescence intensity ratio. Data were obtained from three images per cell, from 10 cells of three independent experiments. Data indicate the mean ± SEM (*P < 0.05). (Scale bar, 5 µm.)