ABSTRACT
Synapse Associated Protein 97 (SAP97), a member of membrane-associated guanylate kinase (MAGUK) protein family, has been involved in the correct targeting and clustering of ionotropic glutamate receptors (iGluRs) at postsynaptic sites. Calcium/calmodulin kinase II (CaMKII) phosphorylates SAP97 on two major sites in vivo; one located in the N-terminal domain (Ser39) and the other in the first postsynaptic density disc large ZO1 (PDZ) domain (Ser232). CaMKII-mediated phosphorylation of SAP97-Ser39 is necessary and sufficient to drive SAP97 to the postsynaptic compartment in cultured hippocampal neurons. CaMKII-dependent phosphorylation of Ser232 disrupts SAP97 interaction with NR2A subunit, thereby regulating synaptic targeting of this NMDA receptor subunit. Here we show by means of phospho-specific antibodies that SAP97-Ser39 phosphorylation represents the driving force to release SAP97/NR2A complex from the endoplasmic reticulum. Ser39 phosphorylation does not interfere with SAP97 capability to bind NR2A. On the contrary, SAP97-Ser232 phosphorylation occurs within the postsynaptic compartment and is responsible for both the disruption of NR2A/SAP97 complex and, consequently, for NR2A insertion in the postsynaptic membrane. Thus, CaMKII-dependent phosphorylation of SAP97 in different time frames and locations within the neurons controls both NR2A trafficking and insertion.
Subject(s)
Adaptor Proteins, Signal Transducing/physiology , Calcium-Calmodulin-Dependent Protein Kinases/pharmacology , Membrane Proteins/physiology , Receptors, N-Methyl-D-Aspartate/metabolism , Animals , Caffeine/pharmacology , Calcium-Calmodulin-Dependent Protein Kinase Type 2 , Cells, Cultured , Cricetinae , Cricetulus , Drug Interactions , Embryo, Mammalian , Endoplasmic Reticulum/drug effects , Endoplasmic Reticulum/metabolism , Excitatory Amino Acid Agonists/pharmacology , Hippocampus/cytology , Immunoprecipitation/methods , In Vitro Techniques , N-Methylaspartate/pharmacology , Neurons/drug effects , Neurons/physiology , Neurons/ultrastructure , Phosphodiesterase Inhibitors/pharmacology , Phosphorylation/drug effects , Protein Transport/drug effects , Rats , Serine/metabolism , Transfection/methodsABSTRACT
Three therapeutic protocols using 3 different bisphosphonate molecules have been evaluated in three groups of women in physiological menopause, whose bone mineral density (BMD) at the ultradistal (UD) or mediodistal (MD) radius showed risk of fracture. Treatment with alendronate (5 mg/day, for 12 months) increases BMD (38 patients, mean age 63 +/- 6 years) by about 5%; treatment with ethidronate (400 mg/day form days, then 1.250 mg/day calcium carbonate for 76 days), repeated 4-times (31 patients, mean age 66 +/- 6 years) did not cause any significant rise in BMD of either UD or MD radius; treatment with clodronate (10 mg/day i.m. for 20 days, a 10-day-interval, followed by 400 mg/day for 60 days) resulted in an insignificant increase (about 4%) in the BMDUD value (8 patients, mean age 62 +/- 5 years). No vertebral fractures nor deformations were observed in any of the 3 groups during treatments. All groups showed an optimal tolerance to the drugs.
