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  • In contrast exposure of CRF a receptors to

    2019-07-10

    In contrast, exposure of CRF2(a) receptors to h/rCRF in living cells produced a strikingly weak translocation of βarrestin2 to the membrane. Specifically, we found a markedly lower magnitude and slower rate of βarrestin2 recruitment to CRF2(a) receptors in response to h/rCRF concentrations as high as 1μM compared to the remarkably more rapid and greater βarrestin2 translocation response induced by 100nM UCN2 and UCN3. This observation agrees with our other findings that h/rCRF induced weak phosphorylation, desensitization, and internalization of the CRF2(a) receptor. A recent study reported, however, similar magnitudes of βarrestin2 translocation to cell surface CRF2(b) receptors activated by 100nM UCN2 or h/rCRF [16]. The 406–414 amino Phosphoramidon Disodium Salt what CRF(2a) receptor and the 430–438 amino-acid CRF(2b) receptor splice variants have identical sequences in their intracellular loops and C-terminal regions [1], [2], [3], [4], [5], [6]. Substantial differences in their distal N-terminal extracellular domains may confer, however, distinct agonist-induced conformations to the two CRF2 receptor splice variants that result in differences in the extent and/or pattern of GRK phosphorylation and βarrestin2 recruitment in response to UCN2 or h/rCRF. Alternatively, activation of CRF2(a) and CRF2(b) receptors by the same ligand may differentially expose phosphorylation-independent βarrestin2 binding motifs. While GRK-mediated GPCR phosphorylation is critical for βarrestin recruitment [13], [14], recent studies have also shown that agonist-induced changes in receptor conformation independent of GRK phosphorylation can also play an important role in the interaction of βarrestin2 with GPCRs including the CRF1 receptor [21], [43], [44].
    Conclusion We have found unique profiles of CRF2(a) receptor phosphorylation, βarrestin2 recruitment, homologous desensitization, and dynamin/clathrin-mediated internalization conferred by binding of urocortins or CRF. Thus, differential desensitizing and internalizing actions of urocortins and CRF may generate agonist-specific regulation of CRF2(a) receptors in brain neurons depending on which ligand is present. Increasing evidence indicates that “biased agonists” promote and stabilize distinct receptor conformations with functional selectivity for specific signal transduction pathways [14], [45]. Previous studies using transfected CHO cells have demonstrated strong ERK signaling by recombinant CRF2(b) receptors activated by UCN2 but not CRF [46]. Our preliminary experiments have detected a large stimulation of Akt phosphorylation by CRF2(a) receptors activated by UCN2 or UCN3 compared to minimal effects from binding CRF [Olivares-Reyes & Hauger, unpublished data]. Rapid and strong recruitment of βarrestin2 to the CRF2(a) receptor during sustained exposure to UCN2 or UCN3 may shift signaling from G protein- to βarrestin2-dependent pathways. CRF2(a) receptors are highly expressed in the hippocampus, the bed nucleus of the stria terminalis, and other regions of the extended amygdala that have been implicated in posttraumatic stress disorder (PTSD) and mood pathophysiology [1], [2], [47]. Furthermore, prolonged severe stress and trauma upregulate expression of limbic CRF2(a) receptors [48], [49]. Stress also promotes CRF2(a) receptor trafficking from the cytoplasm to the cell surface thereby rendering dorsal raphe neurons hyperexcitable (i.e., abnormally high discharge rates) due to excessive CRF2(a) signaling [50]. Importantly, G protein-coupled hypersignaling by brain CRF2(a) receptors has been found to promote startle hyperreactivity, persistent anxiety-like behavior, and contextual fear in mouse models of PTSD [10], [11], [48], [49], [50], [51]. Thus, the failure to rapidly and strongly desensitize limbic CRF2(a) receptors upregulated to abnormally high levels following stress and trauma due to deficient βarrestin2 recruitment may be an important mechanism contributing to PTSD [52]. The following are the supplementary data related to this article.