ABSTRACT
The muscle-specific receptor tyrosine kinase (MuSK) is co-localized with nicotinic acetylcholine receptors (AChRs) in the postsynaptic membrane of the skeletal neuromuscular junction, and is required for all known aspects of postsynaptic differentiation. Studies in vitro have shown that Z(+)-agrin, a nerve-derived proteoglycan, activates MuSK's kinase activity to promote clustering of AChRs and MuSK itself with a cytoplasmic, receptor-associated protein, rapsyn. These studies, however, have used soluble forms of agrin, whereas agrin is cell- or matrix-attached in vivo. We show here that immobilized (particle- or cell-attached) agrin but not soluble agrin is able to aggregate MuSK in the absence of rapsyn and that this aggregation does not require MuSK's kinase activity but does require MuSK's cytoplasmic domain. Moreover, immobilized agrin can promote clustering of AChRs by a mechanism that requires MuSK and rapsyn but does not require MuSK's kinase activity. These results imply that rapsyn and signaling components activated by MuSK kinase may be dispensable for some early aspects of postsynaptic differentiation.
Subject(s)
Muscle Proteins/metabolism , Myoblasts/metabolism , Phosphotransferases/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Cholinergic/metabolism , Agrin/pharmacology , Agrin/physiology , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Embryo, Mammalian , Enzyme Activation , Green Fluorescent Proteins , Humans , Immunohistochemistry/methods , Luminescent Proteins/metabolism , Mice , Mice, Mutant Strains , Muscle Proteins/genetics , Myoblasts/drug effects , Neural Cell Adhesion Molecules/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Receptor Aggregation/drug effects , Receptor Aggregation/physiology , Receptors, Nicotinic/metabolism , Signal Transduction , Transfection/methodsABSTRACT
The pleckstrin homology (PH) domain has been postulated to serve as an anchor for enzymes that operate at a lipid/water interface. To understand further the relationship between the PH domain and enzyme activity, a phospholipase C (PLC) delta1/PH domain enhancement-of-activity mutant was generated. A lysine residue was substituted for glutamic acid in the PH domain of PLC delta1 at position 54 (E54K). Purified native and mutant enzymes were characterized using a phosphatidylinositol 4,5-bisphosphate (PI(4, 5)P2)/dodecyl maltoside mixed micelle assay and kinetics measured according to the dual phospholipid model of Dennis and co-workers (Hendrickson, H. S., and Dennis, E. A. (1984) J. Biol. Chem. 259, 5734-5739; Carmen, G. M., Deems, R. A., and Dennis, E. A. (1995) J. Biol. Chem. 270, 18711-18714). Our results show that both PLC delta1 and E54K bind phosphatidylinositol bisphosphate cooperatively (Hill coefficients, n = 2.2 +/- 0.2 and 2.0 +/- 0.1, respectively). However, E54K shows a dramatically increased rate of (PI(4, 5)P2)-stimulated PI(4,5)P2 hydrolysis (interfacial Vmax for PLC delta1 = 4.9 +/- 0.3 micromol/min/mg and for E54K = 31 +/- 3 micromol/min/mg) as well as PI hydrolysis (Vmax for PLC delta1 = 27 +/- 3.4 nmol/min/mg and for E54K = 95 +/- 12 nmol/min/mg). In the absence of PI(4,5)P2 both native and mutant enzyme hydrolyze PI at similar rates. E54K also has a higher affinity for micellar substrate (equilibrium dissociation constant, Ks = 85 +/- 36 microM for E54K and 210 +/- 48 microM for PLC delta1). Centrifugation binding assays using large unilamelar phospholipid vesicles confirm that E54K binds PI(4,5)P2 with higher affinity than native enzyme. E54K is more active even though the interfacial Michaelis constant (Km) for E54K (0.034 +/- 0.01 mol fraction PI(4,5)P2) is higher than the Km for native enzyme (0.012 +/- 0.002 mol fraction PI(4,5)P2). D-Inositol trisphosphate is less potent at inhibiting E54K PI(4,5)P2 hydrolysis compared with native enzyme. These results demonstrate that a single amino acid substitution in the PH domain of PLC delta1 can dramatically enhance enzyme activity. Additionally, the marked increase in Vmax for E54K argues for a direct role of PH domains in regulating catalysis by allosteric modulation of enzyme structure.
