Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.     

Primary Cilia Reconsidered in the Context of Ciliopathies: Extraciliary and Ciliary Functions of Cilia Proteins Converge on a Polarity theme?

Once dismissed as vestigial organelles, primary cilia have garnered the interest of scientists, given their importance in development/signaling, and for their implication in a new disease category known as ciliopathies. However, many, if not all, “cilia” proteins also have locations/functions outside of the primary cilium. These extraciliary functions can complicate the interpretation of a particular ciliopathy phenotype: it may be a result of defects at the cilium and/or at extraciliary locations, and it could be broadly related to a unifying cellular process for these proteins, such as polarity. Assembly of a cilium has many similarities to the development of other polarized structures. This evolutionarily preserved process for the assembly of polarized cell structures offers a perspective on how the cilium may have evolved. We hypothesize that cilia proteins are critical for cell polarity, and that core polarity proteins may have been specialized to form various cellular protrusions, including primary cilia.

     

Independent ��-Arrestin2 and Gq/Protein Kinase C�� Pathways for ERK Stimulated by Angiotensin Type 1A Receptors in Vascular Smooth Muscle Cells Converge on Transactivation of the Epidermal Growth Factor Receptor

Recent studies in receptor-transfected cell lines have demonstrated that extracellular signal-regulated kinase (ERK) activation by angiotensin type 1A receptor and other G protein-coupled receptors can be mediated by both G protein-dependent and β-arrestin-dependent mechanisms. However, few studies have explored these mechanisms in primary cultured cells expressing endogenous levels of receptors. Accordingly, here we utilized the β-arrestin biased agonist for the angiotensin type 1A receptor, SII-angiotensin (SII), and RNA interference techniques to investigate angiotensin II (ANG)-activated β-arrestin-mediated mitogenic signaling pathways in rat vascular smooth muscle cells. Both ANG and SII induced DNA synthesis via the ERK activation cascade. Even though SII cannot induce calcium influx (G protein activation) after receptor stimulation, it does cause ERK activation, although less robustly than ANG. Activation by both ligands is diminished by depletion of β-arrestin2 by small interfering RNA, although the effect is more complete with SII. ERK activation at early time points but not later time points is strongly inhibited by those protein kinase C inhibitors that can block protein kinase Cζ. Moreover, ANG- and SII-mediated ERK activation require transactivation of the epidermal growth factor receptor via metalloprotease 2/9 and Src kinase. β-Arrestin2 facilitates ANG and SII stimulation of Src-mediated phosphorylation of Tyr-845 on the EGFR, a known site for Src phosphorylation. These studies delineate a convergent mechanism by which G protein-dependent and β-arrestin-dependent pathways can independently mediate ERK-dependent transactivation of the EGFR in vascular smooth muscle cells thus controlling cellular proliferative responses.G protein-coupled receptors, also known as seven transmembrane (7TM)² receptors, control virtually all known physiological processes in mammals (1). The various functions of these receptors are mediated and modulated by three families of proteins, which share the property that they interact virtually universally with the receptors in a strictly stimulus-dependent way (1). These three families of proteins are the heterotrimeric G proteins, the G protein-coupled receptor kinases (GRKs), and the β-arrestins. Activation of the receptors stimulates classical G protein-dependent signaling, often involving regulation of levels of second messengers such as cAMP and diacyglycerol. However, as has been known for many years, interaction of activated receptors with GRKs leading to their phosphorylation, and subsequent interaction with β-arrestins leads to desensitization of G protein signaling.In recent years, however, it has become increasingly clear that the β-arrestin-GRK system is in fact bifunctional (2). Thus, even as it desensitizes G protein signaling by the receptors, it also serves as a signal transduction system in its own right, activating a growing list of signaling pathways. These positive signaling functions are often mediated by the ability of β-arrestin to serve as an adaptor or scaffold molecule, bringing elements of diverse signaling pathways into proximity with one another and the receptors and thereby facilitating their activation. This new paradigm for understanding the previously unrecognized signaling properties of the β-arrestin-GRK system has been explored in a wide variety of transfected cultured cell systems.However, to date, relatively little investigation of these novel signaling pathways has been carried out in primary cell culture systems expressing endogenous levels of 7TM receptors. In seeking such a system in which to characterize and compare β-arrestin and G protein-mediated signaling pathways from a typical 7TM receptor, our attention was drawn to cultured rat vascular smooth muscle cells (VSMCs). Several features of rat VSMCs suggest this to be a relevant system for these purposes. Rat VSMCs express a variety of physiologically important 7TM receptors including the angiotensin II type 1A receptor (AT1R) (3). This receptor has been the focus of extensive study in transfected cell systems with respect to its β-arrestin-mediated signaling to a variety of pathways, most particularly extracellular signal-regulated kinase (ERK). Moreover, the AT1R mediates the physiologically important effects of angiotensin II (ANG) on vascular tone as well as on proliferation and chemotaxis (4, 5). Pathophysiologically, ANG stimulation of this receptor has been implicated in VSMC proliferation and chemotaxis, which are thought to play an important role in such important disease processes as atherosclerosis and restenosis after angioplasty (6, 7). Moreover, a ligand has been characterized [Sar¹,Ile⁴,Ile⁸](SII)-angiotensin (SII), a triply mutated angiotensin octapeptide that, in transfected cell systems, acts as a specific agonist for β-arrestin-mediated signaling, although not activating G protein-mediated signaling (8).Accordingly, in the studies described here, we set out to investigate the characteristics of activation of ERK in rat VSMCs that might be mediated through G protein as well as β-arrestin signaling. The results not only demonstrate the importance of β-arrestin-mediated signaling in ERK-mediated proliferative responses of these cells, but also shed new light on the molecular mechanisms and interrelationships between the β-arrestin and classical G protein-mediated activation of these pathways.