Mechanisms and functions of AT₁ angiotensin receptor internalization

Abstract

The type 1 (AT₁) angiotensin receptor, which mediates the known physiological and pharmacological actions of angiotensin II, activates numerous intracellular signaling pathways and undergoes rapid internalization upon agonist binding. Morphological and biochemical studies have shown that agonist-induced endocytosis of the AT₁ receptor occurs via clathrin-coated pits, and is dependent on two regions in the cytoplasmic tail of the receptor. However, it is independent of G protein activation and signaling, and does not require the conserved NPXXY motif in the seventh transmembrane helix. The dependence of internalization of the AT₁ receptor on a cytoplasmic serine–threonine-rich region that is phosphorylated during agonist stimulation suggests that endocytosis is regulated by phosphorylation of the AT₁ receptor tail. β-Arrestins have been implicated in the desensitization and endocytosis of several G protein-coupled receptors, but the exact nature of the adaptor protein required for association of the AT₁ receptor with clathrin-coated pits, and the role of dynamin in the internalization process, are still controversial. There is increasing evidence for a role of internalization in sustained signal generation from the AT₁ receptor. Several aspects of the mechanisms and specific function of AT₁ receptor internalization, including its precise mode and route of endocytosis, and the potential roles of cytoplasmic and nuclear receptors, remain to be elucidated.

Introduction

The octapeptide hormone, angiotensin II (Ang II), is the major effector molecule of the renin–angiotensin system. Ang II plays a central role in the control of blood pressure through its actions on vascular smooth muscle contractility, aldosterone secretion from adrenal glomerulosa cells, ion transport in renal tubular cells, and dipsogenic responses in the brain [1], [2], [3]. The importance of Ang II in the pathogenesis of hypertension and other cardiovascular diseases has been demonstrated by the therapeutic efficacy of angiotensin-converting enzyme inhibitors and AT₁ receptor (AT₁-R) blockers [3], [4]. The two major subtypes of Ang II receptors expressed in mammalian tissues are the AT₁-R and the AT₂ angiotensin receptor (AT₂-R), which are seven transmembrane receptors with about 30% sequence identity [1], [5]. Despite their similar basic structures, the functions of the AT₁- and AT₂-R subtypes are quite different. The main signal transduction pathway of the AT₁-R, which mediates the major physiological effects of Ang II, is activation of phospholipase C-β1 via the G_q/11 family of G proteins, initiating inositol phosphate responses, Ca²⁺ signal generation and protein kinase C activation [1], [2], [3]. The AT₁-R also activates intracellular signaling pathways that were originally associated with growth factor and cytokine receptors. These include stimulation of tyrosine kinase activity and activation of phospholipase C-γ1, the JAK-STAT pathway, Akt/protein kinase B, and small GTP-binding proteins including Ras and Rho [6], [7]. The activities of these signal transduction pathways are influenced by rapid homologous and heterologous desensitization of the receptor, and long-term down-regulation of receptor expression [8], [9], [10]. On the other hand, the AT₂-R causes growth inhibitory effects, stimulates tyrosine phosphatases, and induces apoptosis [11].

Ang II binding also causes rapid internalization of the AT₁-R [10], [12]. Studies on endogenous and expressed AT₂-Rs have demonstrated that it is an internalization-deficient receptor [13], [14], [15]. Agonist-induced internalization of the AT₄ angiotensin receptor has also been reported recently [16]. The present review will focus on the agonist-induced internalization of the AT₁-R.

Receptor endocytosis is a general feature of plasma membrane receptors, and is mediated by vesicular uptake mechanisms [17], [18], [19]. Most nutrient receptors (e.g., the LDL and transferrin receptors) undergo constitutive (ligand-independent) endocytosis. On the other hand, the internalization of hormone and growth factor receptors is regulated by their specific agonist ligands [17], [18], [19]. Cell surface proteins may internalize by endocytosis via clathrin-coated vesicles [17], caveolae [20], or non-coated vesicles [21]. The role of endocytosis via clathrin-coated vesicles is well established in the recycling of synaptic vesicles, constitutive internalization of nutrient receptors, and agonist-induced internalization of growth factor (e.g., EGF, insulin) receptors [22], [23]. The major mechanism of internalization of GTP-binding protein-coupled receptors (GPCRs) is endocytosis via clathrin-coated vesicles. The agonist-dependence of GPCR internalization suggests that this event is initiated after agonist binding by the isomerization of the receptor to its active conformation [18], [24], [25]. Recent data suggest that endocytosis via clathrin-coated pits is not a homogenous process. Endocytosis of the transferrin receptor and the β₂-adrenergic receptors is mediated largely by separate vesicles, and the β-arrestin content and temperature sensitivity of the formation of these vesicles are different [26]. Inhomogeneities in the mechanism of internalization of GPCRs have also been reported, based on the different sensitivities of these receptors to β-arrestins, dynamin, and GIT1, a GTPase-activating protein of the ADP ribosylation factor family of small GTP-binding proteins [27], [28], [29].

The basic β-arrestin and dynamin-dependent mechanism of internalization of the β₂-adrenergic receptor via clathrin-coated vesicles appears to be utilized by most (but not all) GPCRs (Fig. 1). Endocytosis via clathrin-coated vesicles is preceded by accumulation of the receptors in specialized coated pit regions of the cell surface. The assembly unit of the polygonal lattice on the surface of coated pits is the clathrin triskelion, a three-legged structure consisting of three heavy chains and three tightly associated light chains [22]. Nutrient and growth factor receptors are anchored to clathrin-coated pits by AP2 adaptor proteins [17], [22]. In the case of GPCRs β-arrestins have been proposed to serve as adaptors that connect the activated receptor to clathrin [30], [31]. Coated pits invaginate and pinch off to form coated vesicles under the control of dynamin, a self-associating GTPase [19]. The internalized vesicles are then targeted to fuse with endosomes, and the internalized receptors and their ligands either recycle to the cell surface or undergo lysosomal degradation. Small GTP-binding proteins, including rab5, are also involved in the regulation of the early endocytic pathway of these receptors [17], [19].

An additional internalization pathway is mediated by caveolae, which have been implicated in endocytosis of receptors, transcytosis of macromolecules, and potocytosis of small molecules and ions [32], [33]. Caveolae have also been reported to mediate the internalization of several GPCRs [32]. The caveolar structure is a lipid-based microdomain made up of caveolin protein and specific lipids, including cholesterol as a crucial component [32]. Drugs that alter the cholesterol content of the membrane, such as filipin and nystatin, have been used to indicate the role of caveolae in internalization pathways. The caveolar microdomains can accumulate receptors and signal transduction molecules, and are generally believed to have a role in organization of signaling complexes. Caveolae are morphologically distinct from clathrin-coated vesicles, but their formation has also been reported to be dynamin-dependent [34], [35].

Endocytosis of GPCRs via non-coated vesicles that are distinct from caveolae has also been suggested, based upon the internalization of muscarinic [36] and β₂-adrenergic [37] receptors via non-coated vesicles, but the nature and endocytic mechanism of these vesicles have yet to be elucidated.