Hongwei Qian

Agonist-induced phosphorylation of the angiotensin II (AT(1A)) receptor requires generation of a conformation that is distinct from the inositol phosphate-signaling state

G protein-coupled receptors are thought to isomerize between distinct inactive and active conformations, an idea supported by receptor mutations that induce constitutive (agonist-independent) activation. The agonist-promoted active state initiates signaling and, presumably, is then phosphorylated and internalized to terminate the signal. In this study, we examined the phosphorylation and internalization of wild type and constitutively active mutants (N111A and N111G) of the type 1 (AT1A) angiotensin II receptor. Cells expressing these receptors were stimulated with angiotensin II (AngII) and [Sar1,Ile4,Ile8]AngII, an analog that only activates signaling through the constitutive receptors. Wild type AT1A receptors displayed a basal level of phosphorylation, which was stimulated by AngII. Unexpectedly, the constitutively active AT1A receptors did not exhibit an increase in basal phosphorylation nor was phosphorylation enhanced by AngII stimulation. Phosphorylation of the constitutively active receptors was unaffected by pretreatment with the non-peptide AT1 receptor inverse agonist, EXP3174, and was not stimulated by the selective ligand, [Sar1,Ile4,Ile8]AngII. Paradoxically, [Sar1,Ile4,Ile8]AngII produced a robust (∼85% of AngII), dose-dependent phosphorylation of the wild type AT1A receptor at sites in the carboxyl terminus similar to those phosphorylated by AngII. Moreover, internalization of both wild type and constitutive receptors was induced by AngII, but not [Sar1,Ile4,Ile8]AngII, providing a differentiation between the phosphorylated and internalized states. These data suggest that the AT1A receptor can attain a conformation for phosphorylation without going through the conformation required for inositol phosphate signaling and provide evidence for a transition of the receptor through multiple states, each associated with separate stages of receptor activation and regulation. Separate transition states may be a common paradigm for G protein-coupled receptors.

Caveolin Interacts with the Angiotensin II Type 1 Receptor during Exocytic Transport but Not at the Plasma Membrane

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R·caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.

Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin

Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.

Elevated expression of activins promotes muscle wasting and cachexia

In models of cancer cachexia, inhibiting type IIB activin receptors (ActRIIBs) reverse muscle wasting and prolongs survival, even with continued tumor growth. ActRIIB mediates signaling of numerous TGF‐β proteins; of these, we demonstrate that activins are the most potent negative regulators of muscle mass. To determine whether activin signaling in the absence of tumor‐derived factors induces cachexia, we used recombinant serotype 6 adeno‐associated virus (rAAV6) vectors to increase circulating activin A levels in C57BL/6 mice. While mice injected with control vector gained ~10% of their starting body mass (3.8±0.4 g) over 10 wk, mice injected with increasing doses of rAAV6:activin A exhibited weight loss in a dose‐dependent manner, to a maximum of –12.4% (–4.2±1.1 g). These reductions in body mass in rAAV6:activin‐injected mice correlated inversely with elevated serum activin A levels (7‐ to 24‐fold). Mechanistically, we show that activin A reduces muscle mass and function by stimulating the ActRIIB pathway, leading to deleterious consequences, including increased transcription of atrophy‐related ubiquitin ligases, decreased Akt/mTOR‐mediated protein synthesis, and a profibrotic response. Critically, we demonstrate that the muscle wasting and fibrosis that ensues in response to excessive activin levels is fully reversible. These findings highlight the therapeutic potential of targeting activins in cachexia.—Chen, J. L., Walton, K. L., Winbanks, C. E., Murphy, K. T., Thomson, R. E., Makanji, Y., Qian, H., Lynch, G. S., Harrison, C. A., Gregorevic, P. Elevated expression of activins promotes muscle wasting and cachexia. FASEB J. 28, 28–1711 (1723). www.fasebj.org

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass

The BMP signaling pathway promotes muscle growth and inhibits muscle wasting via SMAD1/5-dependent signaling.

Type 1 angiotensin receptor pharmacology: Signaling beyond G proteins

Abstract Drugs that inhibit the production of angiotensin II (AngII) or its access to the type 1 angiotensin receptor (AT1R) are prescribed to alleviate high blood pressure and its cardiovascular complications. Accordingly, much research has focused on the molecular pharmacology of AT1R activation and signaling. An emerging theme is that the AT1R generates G protein dependent as well as independent signals and that these transduction systems separately contribute to AT1R biology in health and disease. Regulatory molecules termed arrestins are central to this process as is the capacity of AT1R to crosstalk with other receptor systems, such as the widely studied transactivation of growth factor receptors. AT1R function can also be modulated by polymorphisms in the AGTR gene, which may significantly alter receptor expression and function; a capacity of the receptor to dimerize/oligomerize with altered pharmacology; and by the cellular environment in which the receptor resides. Together, these aspects of the AT1R “flavour” the response to angiotensin; they may also contribute to disease, determine the efficacy of current drugs and offer a unique opportunity to develop new therapeutics that antagonize only selective facets of AT1R function.

Casein kinase II sites in the intracellular C-terminal domain of the thyrotropin-releasing hormone receptor and chimeric gonadotropin-releasing hormone receptors contribute to β-arrestin-dependent internalization

We have previously shown that the mammalian gonadotropin-releasing hormone receptor (GnRHR), a unique G-protein-coupled receptor (GPCR) lacking an intracellular carboxyl tail (C-tail), does not follow a β-arrestin-dependent internalization pathway. However, internalization of a chimeric GnRHR with the thyrotropin-releasing hormone receptor (TRHR) C-tail does utilize β-arrestin. Here, we have investigated the sites within the intracellular C-tail domain that are important for conferring β-arrestin-dependent internalization. In contrast to the chimeric GnRHR with a TRHR C-tail, a chimeric GnRHR with the catfish GnRHR C-tail is not β-arrestin-dependent. Sequence comparisons between these chimeric receptors show three consensus phosphorylation sites for casein kinase II (CKII) in the TRHR C-tail but none in the catfish GnRHR C-tail. We thus investigated a role for CKII sites in determining GPCR internalization via β-arrestin. Sequential introduction of three CKII sites into the chimera with the catfish C-tail (H354D,A366E,G371D) resulted in a change in the pattern of receptor phosphorylation and β-arrestin-dependence, which only occurred when all three sites were introduced. Conversely, mutation of the putative CKII sites (T365A,T371A,S383A) in the C-tail of a β-arrestin-sensitive GPCR, the TRHR, resulted in decreased receptor phosphorylation and a loss of β-arrestin-dependence. Mutation of all three CKII sites was necessary before a loss of β-arrestin-dependence was observed. Visualization of β-arrestin/GFP redistribution confirmed a loss or gain of β-arrestin sensitivity for receptor mutants. Internalization of receptors without C-tail CKII sites was promoted by a phosphorylation-independent β-arrestin mutant (R169E), suggesting that these receptors do not contain the necessary phosphorylation sites required for β-arrestin-dependent internalization. Apigenin, a specific CKII inhibitor, blocked the increase in receptor internalization by β-arrestin, thus providing further support for the involvement of CKII. This study presents evidence of a novel role for C-tail CKII consensus sites in targeting these GPCRs to the β-arrestin-dependent pathway.

Phosphoinositide 3-Kinase p110α Is a Master Regulator of Exercise-Induced Cardioprotection and PI3K Gene Therapy Rescues Cardiac Dysfunction

Background—Numerous molecular and biochemical changes have been linked with the cardioprotective effects of exercise, including increases in antioxidant enzymes, heat shock proteins, and regulators of cardiac myocyte proliferation. However, a master regulator of exercise-induced protection has yet to be identified. Here, we assess whether phosphoinositide 3-kinase (PI3K) p110&agr; is essential for mediating exercise-induced cardioprotection, and if so, whether its activation independent of exercise can restore function of the failing heart. Methods and Results—Cardiac-specific transgenic (Tg) mice with elevated or reduced PI3K(p110&agr;) activity (constitutively active PI3K [caPI3K] and dominant negative PI3K, respectively) and non-Tg controls were subjected to 4 weeks of exercise training followed by 1 week of pressure overload (aortic-banding) to induce pathological remodeling. Aortic-banding in untrained non-Tg controls led to pathological cardiac hypertrophy, depressed systolic function, and lung congestion. This phenotype was attenuated in non-Tg controls that had undergone exercise before aortic-banding. Banded caPI3K mice were protected from pathological remodeling independent of exercise status, whereas exercise provided no protection in banded dominant negative PI3K mice, suggesting that PI3K is necessary for exercise-induced cardioprotection. Tg overexpression of heat shock protein 70 could not rescue the phenotype of banded dominant negative PI3K mice, and deletion of heat shock protein 70 from banded caPI3K mice had no effect. Next, we used a gene therapy approach (recombinant adeno-associated viral vector 6) to deliver caPI3K expression cassettes to hearts of mice with established cardiac dysfunction caused by aortic-banding. Mice treated with recombinant adeno-associated viral 6-caPI3K vectors had improved heart function after 10 weeks. Conclusions—PI3K(p110&agr;) is essential for exercise-induced cardioprotection and delivery of caPI3K vector can improve function of the failing heart.

miR-206 represses hypertrophy of myogenic cells but not muscle fibers via inhibition of HDAC4.

microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass in vivo, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 “sponge,” featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype – an important consideration in relation to the development of therapeutics designed to manipulate microRNA activity in musculature.

Arresting angiotensin type 1 receptors

The type 1 angiotensin (AT(1)) receptor mediates the homeostatic and pathological actions of the peptide hormone, angiotensin II. With regard to the processes that activate and deactivate seven-transmembrane-spanning, G-protein-coupled receptors (GPCRs), AT(1) receptors are among the most widely studied, serving as prototypes for GPCRs that bind and respond to peptide hormones. Arrestins are proteins that bind to activated and phosphorylated GPCRs, terminating initial signals emanating from these receptors, in addition to mediating receptor internalization. New aspects of arrestin function continue to emerge, such as their capacity to act as scaffolds to recruit regulatory and signaling molecules to increase the repertoire of receptor responses. Here, we examine the evidence that arrestins contribute to the signaling, deactivation and trafficking of AT(1) receptors.