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  • br Methods and materials br Results

    2020-08-06


    Methods and materials
    Results
    Discussion The data reported herein establishes that the human CRF2(a) receptor is stringently regulated by a rapid and strong desensitization mechanism following activation by the urocortins or related stresscopin peptides but not CRF. Furthermore, UCN2 and UCN3 at saturating concentrations stimulated similar high levels of CRF2(a) receptor phosphorylation and maximal βarrestin2 recruitment while CRF2(a) receptors homologously desensitized and internalized via the dynamin/clathrin-mediated pathway to a greater degree in response to UCN2 compared to UCN3. In contrast, CRF induced very low levels of CRF2(a) receptor phosphorylation, βarrestin2 recruitment, and internalization. These findings indicate the CRF2(a) receptor adopts distinct conformations depending on the bound agonist that can modulate its βarrestin2 recruitment, subcellular trafficking and functional regulation, perhaps through differences in picketing and fencing of the cell membrane by the Isochlorogenic acid C synthesis cytoskeleton known to be involved in synaptic plasticity [38]. We also discovered that deletion of the βarrestin2 gene in MEF cells significantly upregulated Gs-coupled CRF2(a) receptor signaling activated by UCN2 and unrestrained by rapid homologous desensitization. Likewise, isoproterenol-stimulated cyclic AMP accumulation is abnormally high and prolonged in βarrestin2 knockout MEF cells expressing β2-adrenergic adrenergic receptors [22], and UCN2-induced CRF2(b) receptor desensitization is greatly impaired when βarrestin2 protein expression is knocked down by siRNA in HEK293 cells [16]. Neither protein kinase A nor casein kinase mechanisms, however, contributed to CRF2(a) receptor phosphorylation and desensitization. Overall, our study suggests that the rate and magnitude of homologous desensitization of Gs-coupled signaling by the CRF2(a) receptor is governed by the affinity and selectivity of agonist-induced conformations for triggering GRK-dependent phosphorylation, βarrestin2 translocation, and dynamin/clathrin-dependent internalization. Unlike the CRF1 receptor, the CRF2(a) receptor binds and is activated by agonists with a broad range of potencies. Therefore, we assessed the ability of strong and weak ligands to desensitize retinoblastoma CRF2(a) receptors. Although stresscopin\'s N-terminus is two amino acids longer than the N-terminus of human UCN3, while the N-terminus of stresscopin-related peptide is five amino acids longer than the N-terminus of human UCN2 [23], the robust levels of CRF2(a) receptor desensitization were equivalent when comparing the effects of SCP to UCN3 and SRP to UCN2. Importantly, desensitization of cyclic AMP signaling by CRF2(a) receptors generated by exposure to high-affinity, selective (UCN2, UCN3) or non-selective (UCN1, sauvagine) ligands was strikingly greater than that induced by the low-affinity, non-selective ligand CRF. Moreover, CRF2(a) receptors were only minimally phosphorylated and internalized weakly in response to high CRF concentrations known to saturate the receptor protein in contrast to the high magnitude and rapid rate of CRF2(a) receptor phosphorylation and internalization elicited by the urocortins. Similarly, a recent report has shown that the CRF2(b) receptor, the peripheral CRF2 splice variant primarily expressed in cardiovascular, gastrointestinal, and muscle cells, homologously desensitized and internalized to a substantially greater extent in response to UCN2 compared to CRF in HEK293 cells [16]. Interestingly, activation of the β2-adrenergic receptor by different agonists not only influenced the velocity and extent of GRK-mediated phosphorylation and desensitization but also resulted in different serine and threonine residues being phosphorylated in this receptor\'s C-terminus [39], [40]. Furthermore, a recent fluorescence energy transfer (FRET) study concluded that weaker βarrestin2 recruitment and internalization of the β2-adrenergic receptor activated by norepinephrine compared to stronger regulatory responses induced by epinephrine was a consequence of GRK5-mediated phosphorylation being considerably less for norepinephrine compared to epinephrine potentially resulting in different receptor conformations [41]. Similarly, different serine–threonine phosphorylation “bar codes” on the CRF2(a) receptor activated by urocortins or CRF may generate different conformations that govern the rate and magnitude of βarrestin2 recruitment to cell surface CRF2(a) receptors and their subsequent internalization.