Biased agonism on the type I angiotensin receptor (AT 1 -R) can achieve different outcomes via activation of G protein-dependent and -independent cellular responses. In this study, we investigated whether the biased activation of AT 1 -R can lead to different regulation and intracellular processing of the receptor. We analyzed b-arrestin binding, endocytosis, and subsequent trafficking steps, such as early and late phases of recycling of AT 1 -R in human embryonic kidney 293 cells expressing wild-type or biased mutant receptors in response to different ligands. We used Renilla luciferase-tagged receptors and yellow fluorescent protein-tagged b-arrestin2, Rab5, Rab7, and Rab11 proteins in bioluminescence resonance energy transfer measurements to follow the fate of the receptor after stimulation. We found that not only is the signaling of the receptor different upon using selective ligands, but the fate within the cells is also determined by the type of the stimulation. b-arrestin binding and the internalization kinetics of the angiotensin II-stimulated AT 1 -R differed from those stimulated by the biased agonists. Similarly, angiotensin IIstimulated wild-type AT 1 -R showed differences compared with a biased mutant AT 1 -R (DRY/AAY AT 1 -R) with regards to b-arrestin binding and endocytosis. We found that the differences in the internalization kinetics of the receptor in response to biased agonist stimulation are due to the differences in plasma membrane phosphatidylinositol 4,5-bisphosphate depletion. Moreover, the stability of the b-arrestin binding is a major determinant of the later fate of the internalized AT 1 -R receptor.

The chicken Harderian gland (HG) is densely populated in its subepithelial spaces with plasma cells (PC). These immune cells produce and secrete Ig of the IgA, IgG, and IgM classes. Such Ig secretion into the tears affords the upper respiratory tract with protective antibodies. The immunological role of the HG is quite interesting; yet this gland is a site of unusual PC proliferation. Studies of the gland utilizing bromodeoxyuridine (BrdUrd) incorporation into DNA and propidium iodide (PI) staining of PC DNA have verified previous suggestions in the literature that PC of the chicken HG proliferate. Both isolated PC suspensions and frozen sections of the HG from chicks aged 6 to 9 wk reveal that BrdUrd is incorporated into PC DNA. Furthermore, flow cytometric analysis of PI-stained PC indicates a relatively high percentage of PC in S phase of the cell cycle. Continued studies are examining possible mechanisms controlling proliferation and differentiation of PC in the HG. It is believed that the stromal elements of the HG produce and secrete a factor(s) that influences PC proliferation and differentiation. Isolation and characterization of this influencing factor(s) will allow for the possible systemic application of the factor(s) for enhancement of immune responses.