Cardiac effects of AII - AT(1A) receptor signaling, desensitization, and internalization

Angiotensin II receptors present in cardiomyocytes, nonmyocytes (predominantly fibroblasts), nerve terminals, and the heart vasculature mediate the multiple actions of angiotensin II (AII) in the heart, including modulation of normal and pathophysiological cardiac growth. Although the cellular processes that couple AII receptors (principally the AT1 subtype) to effector responses are not completely understood, recent studies have identified an array of signal transduction pathways activated by AII in cardiac cells. These include: the stimulation of phospholipase C which results in the activation of protein kinase C and the release of calcium from intracellular stores; an enhancement of phosphaditic acid formation; the coupling to soluble tyrosine kinase phosphorylation events; the initiation of the mitogen activated protein kinase (MAPK) cascade; and the induction of the STAT (Signal Transducers and Activators of Transcription) signaling pathway. It is tempting to speculate that these latter responses, which have been previously associated with growth factor signaling pathways, are involved in AII-induced cardiac growth. Interestingly, some of these novel pathways are apparently not under the same strict control imposed upon the more classical signaling pathways. Thus, while AII-induced calcium transients are rapidly (within minutes) desensitized following exposure to AII, the MAP kinase pathway is not, and activation of the STAT pathway requires hours of agonist exposure for maximal induction. These observations support an emerging picture in which the downstream signal transduction pathways of AII receptors are initiated and terminated with a distinct temporal arrangement. This organization allows appropriate rapid responses (e.g. vascular contraction) to transient AII exposure, some of which are rapidly terminated, perhaps for protective reasons, and others not. In contrast, additional responses (e.g. growth) probably require prolonged exposure to agonist

Angiotensin-Ii Receptor Endocytosis Involves 2 Distinct Regions Ef the Cytoplasmic Tail - a Role for Residues On the Hydrophobic Face of a Putative Amphipathic Helix

Following agonist stimulation, many receptors are rapidly internalized from the plasma membrane via a mechanism which presumably involves recognition motifs within the cytoplasmic domains of the receptor. We have previously demonstrated (Thomas, W. G., Thekkumkara, T. J., Motel, T. J., and Baker, K. M. (1995) J. Biol Chem. 270, 207-213) that truncation of the angiotensin II (AT(1A)) receptor, to remove 45 amino acids from the cytoplasmic tail, markedly reduced agonist stimulated receptor endocytosis. In the present study, we have stably and transiently expressed wild type and carboxyl terminus mutated AT(1A) receptors in Chinese hamster ovary cells to identify regions and specific amino acids important for this process. Wild type AT(1A) receptors rapidly internalized (t(1/2) = 2.5 min; Y-max = 76.4%) after AII stimulation. Using AT(1A) receptor mutants, truncated and deleted at the carboxyl terminus, two distinct regions important for internalization were identified: one membrane proximal site between residues 315-329 and another distal to Lys(333), within the terminal 26 amino acids. Point mutations (Y302A, Y312A, L316F, Y319A, and K325A) were performed to identify residues contributing to the membrane proximal site. Mutation of Y302A, Y312A, and K325A had little effect on the rate (t(1/2) = 4.3, 2.8, and 2.8 min) and maximal amount (Y-max = 81.7, 67.8, and 73.5%) of AII induced internalization. In contrast, L316F and Y319A mutations displayed an approximately 2.5-fold reduction in rate (t(1/2) = 6.1 and 6.2 min) and L316F a decreased maximal level (Y-max 38.1 and 71.4%, respectively) compared to wild type. Interestingly, Leu(316) and Tyr(319) are closely aligned within the hydrophobic aspect of a putative amphipathic helix, possibly representing an internalization motif for the AT(1A) receptor. We conclude that the AT(1A) receptor does not use the NPXXY (NPLFY(302)) motif, first described for the beta(2)-adrenergic receptor, to mediate agonist stimulated endocytosis. Rather, two distinct regions of the carboxyl terminus are utilized: one involving hydrophobic and aromatic residues on a putative alpha-helix and another serine/threonine-rich domain

Evidence against a role for protein kinase C in the regulation of the angiotensin II (AT1A) receptor

Three putative protein kinase C phosphorylation sites in the carboxyl-terminal region of the angiotensin II AT(1A) receptor suggest that protein kinase C is involved in the regulation and desensitisation of this receptor. We investigated this possibility by measuring angiotensin II induced Ca2+ transients in cultures of neonatal rat cardiac fibroblasts which express predominantly the angiotensin AT(1A) receptor. Stimulating or inhibiting protein kinase C activity had no effect on angiotensin II stimulated Ca2+ transients. In addition, in situ and in vitro kinase assays revealed that a peptide, corresponding to the region of the angiotensin AT(1A) receptor containing the protein kinase C sites, was a poor substrate for protein kinase C. Thus, a heterologous desensitising role for this kinase on angiotensin AT(1A) receptors in these fibroblasts appears unlikely

Stable expression of a functional rat angiotensin II (AT1A) receptor in CHO-K1 cells: Rapid desensitization by angiotensin II

The octapeptide angiotensin II mediates the physiological actions of the renin-angiotensin system through activation of several angiotensin II receptor subtypes; in particular the AT1. In many tissues, the presence of multiple angiotensin II receptor subtypes, together with a low number of receptors, makes it difficult to study biological responses to physiological concentrations (10-11-10-9 M) of angiotensin II. Also, cultured cells show diminished angiotensin II receptor binding with respect to time in culture and passage number. To address these problems, we expressed the recombinant AT1A receptor in CHO-K1 cells. The stably transfected receptor was characterized using radioligand binding studies and functional coupling to cytosolic free calcium. Radioligand binding of [125I] angiotensin II to the angiotensin II receptor was specific, saturable, reversible and modulated by guanine nucleotides. Like the endogenous AT1A receptor, reported in a variety of tissues, the specific, noncompetitive, nonpeptide AII receptor antagonist, EXP3174, blocked binding of [125I] angiotensin II to the transfected receptor. Scatchard analysis demonstrated that the transfected receptor had a dissociation constant of 1.9 nM with a density of 3.4 pmol/mg protein. An important feature of many of the responses to angiotensin II is the rapid desensitization that occurs following agonist occupancy and the development of tachyphylaxis. In AT1A receptor transfected CHO-K1 cells, angiotensin II (10-9 M) stimulated a rapid increase in cytosolic free calcium that was completely desensitized within 50 sec following receptor occupancy. Agonist induced desensitization was unaffected when receptor internalization was blocked by pretreatment with concanavalin A or incubation at 4°C, and no changes in AT1A receptor affinity or number were observed. Receptor desensitization was also unaffected by inhibition or activation of protein kinase C. Thus, we have established a permanent, high-level transfectant of the AT1A receptor in CHO-K1 cells and have shown that these receptors rapidly desensitize following exposure to physiological concentrations of agonist. The mechanism of rapid desensitization is not related to receptor sequestration, internalization or controlled by PKC phosphorylation. This provides an excellent model for studying AII actions mediated through a specific receptor subtype, at subnanomolar concentrations

Activation of the Stat Pathway by Angiotensin-Ii in T3Cho/at(1A) Cells - Cross-Talk Between Angiotensin-Ii and Interleukin-6 Nuclear Signaling

We recently reported that angiotensin II (AII), acting through the STAT (Signal Transducers and Activators of Transcription) pathway, stimulated a delayed SIF (sis-inducing factor)-like DNA binding activity (maximal at 2-3 h) (Bhat, G. J., Thekkumkara, T. J., Thomas, W. G., Conrad, K. M., and Baker, K. M. (1994) J. Biol. Chem. 269, 31443-31449). Using a cell line transfected with the AT(1A) receptor (T3CHO/AT(1A)), we further characterized the AII-induced SIF response and explored the possible rea sons for the delay in stimulated SIF activity, In cells transfected with a chloramphenicol acetyltransferase reporter plasmid, under the control of a SIE (sis-inducing element), AII markedly stimulated chloramphenicol acetyltransferase activity. The delayed SIF activation by AII was not due to a requirement for the release of other SIF inducing factors into the medium and contrasts with the rapid (5 min) induction elicited by the cytokine, interleukin-6 (IL-6), Interestingly, both agents stimulated tyrosine phosphorylation of Stat92 and predominantly the formation of SIF complex A. We tested the hypothesis that AII initially activated an inhibitory pathway, which was responsible for delaying the maximal SIF stimulation until 2 h, Pretreatment of cells for 15 min with AII resulted in significant inhibition of the IL-6-induced nuclear SIF response (10 min) and Stat92 tyrosine phosphorylation, which was blocked by EXP3174, an AT(1) receptor antagonist. This inhibition was transient with return of the IL-6-induced SIF response at 2 h, suggesting that the delayed maximal activation of SIF by AII occurs following an initial transient inhibitory phase, Pretreatment of cells with phorbol 12-myristate 13-acetate for 15 min, to activate protein kinase C, resulted in inhibition of the IL-6-induced SIF response (10 min). However, down-regulation of protein kinase C activity prevented phorbol 12-myristate 13-acetate, but not AII mediated inhibition of the IL-6-induced SIF response. Although the mechanism is not clear, the results presented in this paper raise the interesting possibility that the activation of SIF/Stat92 by AII is characterized by an initial inhibitory phase, followed by the induction process. The observation that AII and IL-6 utilize similar components of the STAT pathway and that AII can cross-talk with IL-6 signaling through inhibition of IL-6-induced SIF/Stat92, implies a modulatory role for AII in cellular responses to cytokines