Zsuzsanna Gáborik

Differential PI 3-kinase dependence of early and late phases of recycling of the internalized AT1 angiotensin receptor

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.

Mechanisms and functions of AT1 angiotensin receptor internalization

The type 1 (AT(1)) 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 AT(1) 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 AT(1) receptor on a cytoplasmic serine-threonine-rich region that is phosphorylated during agonist stimulation suggests that endocytosis is regulated by phosphorylation of the AT(1) receptor tail. beta-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 AT(1) 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 AT(1) receptor. Several aspects of the mechanisms and specific function of AT(1) receptor internalization, including its precise mode and route of endocytosis, and the potential roles of cytoplasmic and nuclear receptors, remain to be elucidated.

Intracellular trafficking of hormone receptors

Agonist binding stimulates endocytosis of hormone receptors via vesicular uptake mechanisms. Interactions of the intracellular domains of receptors with specific targeting proteins are crucial for sorting of internalized receptor in endosomes. Some receptors are targeted for very rapid (e.g. beta2-adrenergic receptor) or slower (e.g. AT1 angiotensin receptor) recycling pathways, whereas others are targeted to lysosomes for degradation (e.g. EGF receptor or PAR1 protease-activated receptor). This review discusses the mechanisms involved in these processes, which regulate surface receptor expression and set the stage for intracellular signaling of G protein-coupled and growth factor receptors.

Sec14 Homology Domain Targets p50RhoGAP to Endosomes and Provides a Link between Rab and Rho GTPases

Sec14 protein was first identified in Saccharomyces cerevisiae, where it serves as a phosphatidylinositol transfer protein that is essential for the transport of secretory proteins from the Golgi complex. A protein domain homologous to Sec14 was identified in several mammalian proteins that regulates Rho GTPases, including exchange factors and GTPase activating proteins. P50RhoGAP, the first identified GTPase activating protein for Rho GTPases, is composed of a Sec14-like domain and a Rho-GTPase activating protein (GAP) domain. The biological function of its Sec14-like domain is still unknown. Here we show that p50RhoGAP is present on endosomal membranes, where it colocalizes with internalized transferrin receptor. We demonstrate that the Sec14-like domain of P50RhoGAP is responsible for the endosomal targeting of the protein. We also show that overexpression of p50RhoGAP or its Sec14-like domain inhibits transferrin uptake. Furthermore, both P50RhoGAP and its Sec14-like domain show colocalization with small GTPases Rab11 and Rab5. We measured bioluminescence resonance energy transfer between p50RhoGAP and Rab11, indicating that these proteins form molecular complex in vivo on endosomal membranes. The interaction was mediated by the Sec 14-like domain of p50RhoGAP. Our results indicate that Sec14-like domain, which was previously considered as a phospholipid binding module, may have a role in the mediation of protein-protein interactions. We suggest that p50RhoGAP provides a link between Rab and Rho GTPases in the regulation of receptor-mediated endocytosis.

Differential β‐arrestin binding of AT1 and AT2 angiotensin receptors

Agonist stimulation of G protein‐coupled receptors causes receptor activation, phosphorylation, β‐arrestin binding and receptor internalization. Angiotensin II (AngII) causes rapid internalization of the AT1 receptors, whereas AngII‐bound AT2 receptors do not internalize. Although the activation of the rat AT1A receptor with AngII causes translocation of β‐arrestin2 to the receptor, no association of this molecule with the AT2 receptor can be detected after AngII treatment with confocal microscopy or bioluminescence resonance energy transfer. These data demonstrate that the two subtypes of angiotensin receptors have different mechanisms of regulation.

Lys 199 mutation of the human angiotensin type 1 receptor differentially affects the binding of surmountable and insurmountable non-peptide antagonists

Many slow dissociating (insurmountable) non-peptide angiotensin type 1 receptor (AT1) antagonists contain, besides the acidic biphenyltetrazole substructure of losartan, a second acidic group to stabilise antagonist-receptor complexes. To investigate the involved basic amino-acids of the human AT1-receptor, wild-type and mutant receptors were transiently transfected in CHO-K1 cells and characterised by [3H]candesartan binding. Lys199 → Gln substitution decreased the affinity 45-fold for candesartan (95% insurmountable), 18-fold for EXP3174 (70% insurmountable), 10-fold for irbesartan (40% insurmountable) and 5-fold for losartan (surmountable). His256 → Ala substitution had only minor effects. This suggests that Lys199 is important for the tight binding of non-peptide antagonists.

Requirement of membrane-proximal amino acids in the carboxyl-terminal tail for expression of the rat AT1a angiotensin receptor

A series of deletion mutants was created to analyze the function of the membrane‐proximal region of the cytoplasmic tail of the rat type 1a (AT1a) angiotensin receptor. In transiently transfected COS‐7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar1,Ile8]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II‐induced inositol phosphate responses was observed. Alanine substitutions in the region 307–311 identified the highly conserved phenylalanine309 and adjacent lysine residues as significant determinants of AT1a receptor expression.

Structural determinants of agonist-induced signaling and regulation of the angiotensin AT1 receptor.

Angiotensin II (Ang II) regulates aldosterone secretion by stimulating inositol phosphate production and Ca(2+) signaling in adrenal glomerulosa cells via the G(q)-coupled AT(1) receptor, which is rapidly internalized upon agonist binding. Ang II also binds to the heptahelical AT(2) receptor, which neither activates inositol phosphate signaling nor undergoes receptor internalization. The differential behaviors of the AT(1) and AT(2) receptors were analyzed in chimeric angiotensin receptors created by swapping the second (IL2), the third (IL3) intracellular loops and/or the cytoplasmic tail (CT) between these receptors. When transiently expressed in COS-7 cells, the chimeric receptors showed only minor alterations in their ligand binding properties. Measurements of the internalization kinetics and inositol phosphate responses of chimeric AT(1A) receptors indicated that the CT is required for normal receptor internalization, and IL2 is a determinant of G protein activation. In addition, the amino-terminal portion of IL3 is required for both receptor functions. However, only substitution of IL2 impaired Ang II-induced ERK activation, suggesting that alternative mechanisms are responsible for ERK activation in signaling-deficient mutant AT(1) receptors. Substitution of IL2, IL3, or CT of the AT(1A) receptor into the AT(2) receptor sequence did not endow the latter with the ability to internalize or to mediate inositol phosphate signaling responses. These data suggest that the lack of receptor internalization and inositol phosphate signal generation by the AT(2) receptor is a consequence of its different activation mechanism, rather than the inability of its cytoplasmic domains to couple to intracellular effectors.

Role of basic amino acids of the human angiotensin type 1 receptor in the binding of the non-peptide antagonist candesartan

To explain the insurmountable/long-lasting binding of biphenyltetrazole-containing AT1-receptor antagonists such as candesartan, to the human angiotensin II type 1-receptor, a model is proposed in which the basic amino acids Lys199 and Arg 167 of the receptor interact respectively with the carboxylate and the tetrazole group of the antagonists. To validate this model, we have investigated the impact of substitution of Lys199 by Ala or Gln and of Arg167 by Ala on the binding properties of [3H]candesartan and on competition binding by candesartan, EXP3174, irbesartan, losartan, angiotensin II (Ang II) and [Sar1-Ile8]angiotensin. Our results indicate that both amino acids play an important role in the AT1-receptor ligand binding. Whereas the negative charge of Lys 199 is involved in an ionic bond with the end-standing carboxylate group of the peptide ligands, its polarity also contributes to the non-peptide antagonist binding. Substitution of Arg167 by Ala completely abolished [3H]Ang II, as well as [3H] candesartan, binding. Whereas these results are in line with the proposed model, it cannot be excluded that both amino acid residues are important for the structural integrity of the AT1-receptor with respect to its ligand binding properties.

Review: Structural requirements for signalling and regulation of AT1-receptors

JRAAS 2001;2 (suppl 1):S16-S23 Introduction Angiotensin II (Ang II) is an octapeptide hormone that binds to two distinct heptahelical receptors, the AT1and AT2-receptors. The AT1-receptor, which has structurally and functionally similar AT1A and AT1B subtypes in rodents, mediates the crucial physiological and pathophysiological actions of Ang II on blood pressure regulation, vascular tone and salt-water balance. Early studies on the therapeutic use of peptide angiotensin receptor antagonists were unsuccessful owing to the partial agonist activity of these compounds. Since the discovery of non-peptide angiotensin receptor blockers, which selectively recognise the AT1-receptor and have no agonist activity, the AT1receptor has become a major therapeutic target in the management of hypertension and other cardiovascular diseases. Binding of Ang II to the AT1-receptor causes activation of phospholipase C via the Gq/11 family of G proteins, initiating inositol phosphate responses, Ca signal generation and protein kinase C activation. This second messenger system is the main intracellular pathway that mediates the physiological effects of Ang II via AT1receptor activation.These include smooth muscle contraction,aldosterone secretion,and the control of ion transport in renal tubule cells. More recent studies have revealed that Ang II has important effects on the normal and pathological growth of its target cells, and on the remodelling of cardiac and vascular cells. Some of these effects are attributable to Ang II-induced calcium signalling, but it has become evident that multiple forms of signal transduction can participate in such responses. These pathways include activation of receptor and non-receptor protein tyrosine kinases, stimulation of the signal transducers and activators of transcription (STAT) pathway, and small G proteins, including Ras, Rho, Rac, and induction of the expression of other important regulatory enzymes, such as phospholipase D, phospholipase A2, and NAD(P)H oxidase. The tyrosine kinases activated by Ang II include c-Src, focal adhesion kinase (FAK), Ca-dependent tyrosine kinases, such as proline-rich tyrosine kinase 2 (PYK2)/cell adhesion kinase β(CAKβ), Janus kinases (JAK2 and TYK2), and receptor tyrosine kinases, including the epidermal growth factor (EGF) receptor, the plateletderived growth factor (PDGF) receptor, the insulinlike growth factor-I (IGF-I) receptor and Axl. Another important aspect of AT1-receptor activation is the agonist-induced internalisation of the ligand-receptor complex. Although other pathways have also been implicated, internalisation of the AT1-receptor occurs predominantly by endocytosis via clathrin-coated pits. Most of the available data indicate that internalisation of G protein-coupled receptors (GPCRs) is important for the regulation of receptor sensitivity in at least two ways. First, it reduces the number of available cell surface receptors, and secondly, it facilitates resensitisation of plasma membrane receptors that have been desensitised by GPCR kinase (GRK)-mediated phosphorylation. It has been suggested that dephosphorylation of GPCRs (e.g., the β2-adrenergic receptor) occurs within the endosomes after receptor endocytosis, and that the subsequent recycling of the resensitised receptor to the cell surface maintains signal generation. In addition to its role in the regulation of receptor sensitivity, the internalisation process has been proposed to contribute to the initiation of multiple intracellular signalling pathways. For example, internalisation of the receptor appears to be required for extracellular signal-regulated kinase (ERK) activation by the β2-adrenergic receptor and certain other GPCRs. However, ERK activation by many GPCRs is independent of receptor internalisation. In addition to advances in understanding the complexity of the AT1-receptor-activated signal transduction pathways, recent studies have begun to identify the molecular mechanisms that occur during receptor activation. Improved understanding of the structural requirements for the operation of signal transduction pathways should provide clues about the sequence of events that occur during Ang II action. The results of these studies are summarised in this paper.