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 (CRF 2 R). CRF 2 R 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 CRF 2 R, exerting an escort protein role. We analyzed the colocalization of CRF-BP and CRF 2 R in cultured rat mesencephalic neurons, and the localization and interaction of heterologous expressed CRF-BP and CRF 2α R in yeast, human embryonic kidney 293, and rat pheochromocytoma 12 cells. Our results showed that CRF-BP and CRF 2 R 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 CRF 2α R in the endoplasmic reticulum. In contrast, CRF-BP and CRF 2α R colocalized when both proteins are coexpressed. Here we show that CRF-BP physically interacts with the CRF 2α R but not the CRF 2β R isoform, increasing CRF 2α R on the cell surface. Thus, CRF-BP emerges as a GPCR escort protein increasing the understanding of GPCR trafficking.T he corticotropin releasing factor (CRF) system plays a key role in the response and adaptation to stressful stimuli (1, 2) and in the interaction between stress and addiction (3). The CRF system acts on the hypothalamic-pituitary-adrenal axis (4, 5) and in different brain regions (1, 6). The CRF system includes four peptides, CRF type 1 (CRF 1 R) and type 2 (CRF 2 R), G protein-coupled receptors (GPCRs) (6-8), and CRF binding protein (CRF-BP) (9). CRF-BP was described as a circulating polypeptide in pregnant women (10, 11). CRF-BP binds CRF and urocortin with high affinity (12). Different functions have been proposed for CRF-BP (13). On one hand, CRF-BP exerts an inhibitory role by sequestering CRF peptide (9, 14-16). On other hand, a facilitatory role of CRF-BP on CRF-dependent neuronal plasticity depending on CRF 2 R in the rat ventral tegmental area (VTA) has been described (17, 18). Recently, it has been shown that CRF-BP and CRF 2 R are important for ethanol binge drinking behavior (19). The anatomical evidence showing that CRF-BP has different subcellular distribution depending on the neuronal context (20) further supports several roles for CRF-BP.Three isoforms of CRF 2 R have been reported, the α isoform being the most expressed in the brain (21). CRF 2α R is localized intracellularly in neurons of the rat dorsal raphe nucleus and that exposure to acute (22) and repeated stress (23) increases its presence in the plasma membrane. CRF 2α R overexpressed in human embryonic kidney (HEK293T) cells is associated with the endoplasmic reticulum (ER) (24). Schulz et al. (25) showed that CRF 2α R is retained in...

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“…CRF1 cell surface trafficking is modulated by novel interactions with postsynaptic density 95 (PSD 95) and with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) [28, 29]. CRF binding protein (CRF-BP) acts to further regulate this system through sequestering CRF and through increasing CRF 2a cell surface levels [30]. …”

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