Agonist-induced endocytosis and processing of the G protein–coupled AT₁ angiotensin II (Ang II) receptor (AT₁R) was studied in HEK 293 cells expressing green fluorescent protein (GFP)– or hemagglutinin epitope–tagged forms of the receptor. After stimulation with Ang II, the receptor and its ligand colocalized with Rab5–GFP and Rab4–GFP in early endosomes, and subsequently with Rab11–GFP in pericentriolar recycling endosomes. Inhibition of phosphatidylinositol (PI) 3-kinase by wortmannin (WT) or LY294002 caused the formation of large endosomal vesicles of heterogeneous Rab composition, containing the ligand–receptor complex in their limiting membranes and in small associated vesicular structures. In contrast to Alexa^®–transferrin, which was mainly found in small vesicles associated with the outside of large vesicles in WT-treated cells, rhodamine–Ang II was also segregated into small internal vesicles. In cells labeled with ¹²⁵I-Ang II, WT treatment did not impair the rate of receptor endocytosis, but significantly reduced the initial phase of receptor recycling without affecting its slow component. Similarly, WT inhibited the early, but not the slow, component of the recovery of AT₁R at the cell surface after termination of Ang II stimulation. These data indicate that internalized AT₁ receptors are processed via vesicles that resemble multivesicular bodies, and recycle to the cell surface by a rapid PI 3-kinase–dependent recycling route, as well as by a slower pathway that is less sensitive to PI 3-kinase inhibitors.

Stimulation of the angiotensin II (Ang II) type 1 receptor (AT1-R) causes phosphorylation of extracellularly regulated kinases 1 and 2 (ERK1/2) via epidermal growth factor receptor (EGF-R) transactivation-dependent or -independent pathways in Ang II target cells. Here we examined the mechanisms involved in agonist-induced EGF-R transactivation and subsequent ERK1/2 phosphorylation in clone 9 (C9) hepatocytes, which express endogenous AT1-R, and COS-7 and human embryonic kidney (HEK) 293 cells transfected with the AT1-R. Ang II-induced ERK1/2 activation was attenuated by inhibition of Src kinase and of matrix metalloproteinases (MMPs) in C9 and COS-7 cells, but not in HEK 293 cells. Agonist-mediated MMP activation in C9 cells led to shedding of heparin-binding EGF (HB-EGF) and stimulation of ERK1/2 phosphorylation. Blockade of HB-EGF action by neutralizing antibody or its selective inhibitor, CRM197, attenuated ERK1/2 activation by Ang II. Consistent with its agonist action, HB-EGF stimulation of these cells caused marked phosphorylation of the EGF-R and its adapter molecule, Shc, as well as ERK1/2 and its dependent protein, p90 ribosomal S6 kinase, in a manner similar to that elicited by Ang II or EGF. Although the Tyr319 residue of the AT1-R has been proposed to be an essential regulator of EGF-R transactivation, stimulation of wild-type and mutant (Y319F) AT1-R expressed in COS-7 cells caused EGF-R transactivation and subsequent ERK1/2 phosphorylation through release of HB-EGF in a Src-dependent manner. In contrast, the noninvolvement of MMPs in HEK 293 cells, which may reflect the absence of Src activation by Ang II, was associated with lack of transactivation of the EGF-R. These data demonstrate that the individual actions of Ang II on EGF-R transactivation in specific cell types are related to differential involvement of MMP-dependent HB-EGF release.

The agonist-induced phosphorylation sites of the rat AT_1aangiotensin receptor were analyzed using epitope-tagged mutant receptors expressed in Cos-7 cells. Angiotensin II-stimulated receptor phosphorylation was unaffected by truncation of the cytoplasmic tail of the receptor at Ser342 (Δ342) but was abolished by truncation at Ser325 (Δ325). Truncation at Ser335 (Δ335), or double-point mutations of Ser335 and Thr336 to alanine (ST-AA), reduced receptor phosphorylation by ∼50%, indicating that in addition to Ser335 and/or Thr336, amino acids within the Ser326-Thr332 segment are also phosphorylated. Agonist-induced phosphorylation of the ST-AA and Δ335 receptors was partially inhibited by staurosporine, suggesting that the single protein kinase C consensus site in the Ser326-Thr332 segment (Ser331) is phosphorylated. The impairment of receptor phosphorylation was broadly correlated with the attenuation of agonist-induced internalization rates (Δ325 < Δ335 < ST-AA < Δ342 < wild-type) and with the increasing rank order of magnitude of inositol phosphate production normalized to an equal number of receptors (Δ325 > Δ335 > ST-AA = Δ342 > wild-type). These results demonstrate that agonist-induced phosphorylation of the AT_1a receptor is confined to an 11-amino-acid serine/threonine-rich segment of its carboxyl-terminal cytoplasmic tail and implicate this region in the mechanisms of receptor internalization and desensitization.