Luminal ANG II is internalized as a complex with AT₁R/AT₂R heterodimers to target endoplasmic reticulum in LLC-PK₁...

ANG II has many biological effects in renal physiology, particularly in Ca²⁺ handling in the regulation of fluid and solute reabsorption. It involves the systemic endocrine renin-angiotensin system (RAS), but tissue and intracrine ANG II are also known. We have shown that ANG II induces heterodimerization of its AT₁ and AT₂ receptors (AT₁R and AT₂R) to stimulate sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA) activity. Thus, we investigated whether ANG II-AT₁R/AT₂R complex is formed and internalized, and also examined the intracellular localization of this complex to determine how its effect might be exerted on renal intracrine RAS. Living cell imaging of LLC-PK₁ cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with AT₁R/AT₂R heterodimers as a complex in a microtubule-dependent and clathrin-independent manner, since colchicine-but not Pitstop2-blocked this process. This result was confirmed by an increase of β-arrestin phosphorylation after ANG II treatment, clathrin-mediated endocytosis being dependent on dephosphorylation of β-arrestin. Internalized ANG II colocalized with an endoplasmic reticulum (ER) marker and increased levels of AT₁R, AT₂R, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT₁/AT₂ complex to target ER, where it might trigger intracellular Ca²⁺ responses.