Unlike the CRF receptor the CRF a

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 Sulforaphane 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. Our study using real-time confocal microscopy in live cells provides the first evidence that the human CRF2(a) receptor activated by UCN2 or UCN3 rapidly and strongly recruits GFP-labeled βarrestin2 to the plasma membrane where βarrestin2 proteins colocalize with CRF2(a) receptor proteins. Similar to the CRF1 receptor [21], [42], βarrestin2 dissociates from the activated CRF2(a) receptor at or near the plasma membrane and fails to internalize with the CRF2(a) receptor as a unit into endocytic vesicles. Therefore, both CRF receptor isoforms exhibit a “class A” GPCR–arrestin interaction. A recent study using immunohistochemistry in fixed HEK293 cells found that the peripherally expressed CRF2(b) receptor also exhibits a “class A” interaction with βarrestins [16]. To quantify differences in βarrestin2–GFP recruitment stimulated by the urocortins, we transiently expressed the CRF2(a) receptors in U2OS cells, which allow for accurate and sensitive quantitation due to their superior adherent and morphological properties [21]. Although saturating concentrations of UCN2 and UCN3 stimulated similar high levels of CRF2(a) receptor phosphorylation and maximal βarrestin2 recruitment, we found the βarrestin2 recruitment process to be more sensitive when CRF2(a) receptors were activated by UCN2 compared to UCN3, in accordance with the more rapid desensitizing action of UCN2 compared to UCN3. Since UCN3 induces less CRF2(a) receptor desensitization and internalization than that resulting from UCN2, the higher residual level of phosphorylated membrane CRF2(a) receptors during UCN3 exposure may interact with recruited βarrestin2 remaining near the membrane to promote UCN3-specific βarrestin2-mediated signaling and other cellular events.