Luminal ANG II is internalized as a complex with AT₁R/AT₂R heterodimers to target endoplasmic reticulum in LLC-PK₁cells

Abstract

ANG II has many biological effects in renal physiology, particularly in Ca 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 AT and AT receptors (ATR and ATR) to stimulate sarco(endo)plasmic reticulum Ca-ATPase (SERCA) activity. Thus, we investigated whether ANG II-ATR/ATR 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-PK cells, quantification of extracellular ANG II, and use of the receptor antagonists, losartan and PD123319, showed that ANG II is internalized with ATR/ATR 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 ATR, ATR, and PKCα in ER-enriched membrane fractions. This novel evidence suggests the internalization of an ANG II-AT/AT complex to target ER, where it might trigger intracellular Ca responses.

Critical role of angiotensin II type 2 receptors in the control of mitochondrial and cardiac function in angiotensin II-preconditioned rat hearts

“…Of note, plasma membrane AT2-Rs working through tyrosine and serine/threonine phosphatases, modulate protein kinases involved in AT1-R-dependent growth and proliferation [51]. Besides, AT2-Rs could target AT1-Rs to the mitochondria through AT1-R/AT-2R heterodimers as shown in the endoplasmic reticulum of LCC-PK1 cells [15]. The mechanisms involved in AT1-R modulation by AT2-R in mitochondria could be similar but acting on the transcription or synthesis of AT1-Rs and their translocation or targeting to the mitochondria.…”

“…During ischemia, hearts were immersed in KHS buffer maintained at 37°C. The concentrations of losartan and PD used in this study were chosen from previous work with cultured cells [15], vasculature [5], and isolated hearts [17,30,41,45]. A diagram of perfusion protocols is illustrated in Figure 1.…”

No answer to the lack of specificity: mouse monoclonal antibody targeting the angiotensin II type 1 receptor AT1 fails to recognize its target

“…To our knowledge, this is the first report questioning the specificity of Santa Cruz monoclonal AT1 receptor antibody sc-57036. Already used in 10 publications (including three in 2017 and one in 2018) (Filice et al 2017;Yim et al 2017;Ferrão et al 2017;Sinphitukkul et al 2018), this antibody that lacks specificity could lead to false positive staining on tissue or primary cell culture and therefore misinterpretations.…”

Angiotensin Receptors Heterodimerization and Trafficking: How Much Do They Influence Their Biological Function?

“…In transfected HeLa cells, AT2R inhibits the signaling of AT1R that is induced by the ligand through a pathway dependent on protein kinase C (PKC) activation, and this effect results from constitutive AT1R-AT2R heteromerization (Inuzuka et al, 2016). In proximal tubule cells from pig kidney (LLC-PK1 cells), it has been shown that Ang II internalized together with AT1R-AT2R heteromers forming a complex in a process that was dependent on microtubules but not on clathrin to target endoplasmic reticulum, where it might increase sarco(endo) plasmic reticulum calcium ATPase activity and calcium levels (Ferrao et al, 2017). AT1R endocytosis is also influenced by interaction with other receptors.…”