Transmissible spongiform encephalopathies (TSEs) are lethal, infectious disorders of the mammalian nervous system. A TSE hallmark is the conversion of the cellular protein PrP C to disease-associated PrP Sc (named for scrapie, the first known TSE). PrP C is protease-sensitive, monomeric, detergent soluble, and primarily ␣-helical; PrP Sc is protease-resistant, polymerized, detergent insoluble, and rich in ␤-sheet. The ''protein-only'' hypothesis posits that PrP Sc is the infectious TSE agent that directly converts host-encoded PrP C to fresh PrP Sc , harming neurons and creating new agents of infection. To gain insight on the conformational transitions of PrP, we tested the ability of several protein chaperones, which supervise the conformational transitions of proteins in diverse ways, to affect conversion of PrP C to its protease-resistant state. None affected conversion in the absence of pre-existing PrP Sc . In its presence, only two, GroEL and Hsp104 (heat shock protein 104), significantly affected conversion. Both promoted it, but the reaction characteristics of conversions with the two chaperones were distinct. In contrast, chemical chaperones inhibited conversion. Our findings provide new mechanistic insights into nature of PrP conversions, and provide a new set of tools for studying the process underlying TSE pathogenesis.

G protein-coupled receptor-mediated signaling is attenuated by a process referred to as desensitization, wherein agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinases (GRKs) is proposed to be a key initial event. However, mechanisms that activate GRKs are not fully understood. In one scenario, beta gamma-subunits of G proteins (G beta gamma) activate certain GRKs (beta-adrenergic receptor kinases 1 and 2, or GRK2 and GRK3), via a pleckstrin homology domain in the COOH terminus. This interaction has been proposed to translocate cytosolic beta-adrenergic receptor kinases (beta ARKs) to the plasma membrane and facilitate interaction with receptor substrates. Here, we report a novel finding that membrane lipids modulate beta ARK activity in vitro in a manner that is analogous and competitive with G beta gamma. Several lipids, including phosphatidylserine (PS), stimulated, whereas phosphatidylinositol 4,5-bisphosphate inhibited, the ability of these GRKs to phosphorylate agonist-occupied m2 muscarinic acetylcholine receptors. Furthermore, both PS and phosphatidylinositol 4,5-bisphosphate specifically bound to beta ARK1, whereas phosphatidylcholine, a lipid that did not modulate beta ARK activity, did not bind to beta ARK1. The lipid regulation of beta ARKs did not occur via a modulation of its autophosphorylation state. PS- and G beta gamma-mediated stimulation of beta ARK1 was compared and found strikingly similar; moreover, their effects together were not additive (except at initial stages of reaction), which suggests that PS and G beta gamma employed a common interaction and activation mechanism with the kinase. The effects of these lipids were prevented by two well known G beta gamma-binding proteins, phosducin and GST-beta ARK-(466-689) fusion protein, suggesting that the G beta gamma-binding domain (possibly the pleckstrin homology domain) of the GRKs is also a site for lipid:protein interaction. We submit the intriguing possibility that both lipids and G proteins co-regulate the function of GRKs.

G protein-coupled receptor kinases (GRKs) mediate agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) and initiate homologous receptor desensitization. Previously, we reported that charged phospholipids directly interacted with the two GRK isoforms, GRK2 and GKR3, via a pleckstrin homology (PH) domain to regulate GRK activity (DebBurman, S. K., Ptasienski, J., Boetticher, E., Lomasney, J. W., Benovic, J. L., and Hosey, M. M. (1995) J. Biol. Chem. 270: 5742-5747). Here, evidence is provided to support the hypothesis that charged phospholipids are required for agonist-dependent phosphorylation of receptors by GRK2. In the absence of charged phospholipids, the purified human m2 muscarinic acetylcholine receptor (hm2mAChR) reconstituted in pure phosphatidylcholine vesicles or in a noninhibitory detergent was not a substrate for GRK2. However, these receptor preparations were stoichiometrically phosphorylated in an agonist-dependent manner upon addition of charged phospholipids. The known ability of G protein ␤␥ subunits to stimulate mAChR phosphorylation also was found to be absolutely dependent on the presence of charged phospholipids, including phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Phospholipids also regulated GRK-mediated phosphorylation of casein, a nonreceptor-soluble substrate. Among lipids tested, lipid inositol phosphates, PIP 2 and phosphatidylinositol 4-monophosphate, were found to be the most potent activators of GRK2 and were the only lipids that regulated GRK2 in a complex biphasic manner. At low M concentrations, PIP 2 activated GRK2 via an interaction with the GRK pleckstrin homology domain; however, at high M concentrations, PIP 2 inhibited GRK2, apparently via another mechanism. PIP 2 -mediated inhibition could be partly relieved by increasing ATP. The results support the hypothesis that GRK2 is a lipid-dependent protein kinase that requires charged phospholipids for enzyme activation, for regulation by G ␤␥ subunits, and potentially for membrane association.Regulatory mechanisms that control biological responses involve both the amplification and desensitization of cellular signaling (1). For G protein-coupled receptors (GPRs), 1 which represent a diverse superfamily of membrane receptors, agonist-induced phosphorylation of the receptors is thought to be a critical event in the initiation of receptor desensitization (2-4). G protein-coupled receptor kinases (GRKs) are extremely selective protein kinases that phosphorylate specifically only agonist-occupied active form of GPRs (5). A variety of approaches including reconstitution studies in vitro (6 -9), overexpression of protein kinases (10), use of protein kinase inhibitors (11, 12), receptor mutagenesis (13-15), antisense GRK knockout (16), dominant-negative inhibition of GRK (15,17,18), and transgenic mice that overexpress a GRK-selective inhibitory peptide (19), strongly suggest that GRKs mediate agonist-induced phosphorylation of GPRs and trigger homologous desensitization of GPRs.Only recently have studies elucid...

Facilitating not only the mastery of sophisticated subject matter, but also the development of process skills is an ongoing challenge in teaching any introductory undergraduate course. To accomplish this goal in a sophomore-level introductory cell biology course, I require students to work in groups and complete several mock experiential research projects that imitate the professional activities of the scientific community. I designed these projects as a way to promote process skill development within content-rich pedagogy and to connect text-based and laboratory-based learning with the world of contemporary research. First, students become familiar with one primary article from a leading peer-reviewed journal, which they discuss by means of PowerPointbased journal clubs and journalism reports highlighting public relevance. Second, relying mostly on primary articles, they investigate the molecular basis of a disease, compose reviews for an inhouse journal, and present seminars in a public symposium. Last, students author primary articles detailing investigative experiments conducted in the lab. This curriculum has been successful in both quarter-based and semester-based institutions. Student attitudes toward their learning were assessed quantitatively with course surveys. Students consistently reported that these projects significantly lowered barriers to primary literature, improved research-associated skills, strengthened traditional pedagogy, and helped accomplish course objectives. Such approaches are widely suited for instructors seeking to integrate process with content in their courses.