CYCLIC-AMP AND SYNAPTIC ACTIVITY-DEPENDENT PHOSPHORYLATION OF AMPA-PREFERRING GLUTAMATE RECEPTORS

Several studies have suggested that the function of glutamate receptor channels can be regulated by protein phosphorylation. Furthermore, a basal level of phosphorylation may be necessary to maintain receptor f unction. Little is known, however, about the phosphorylation state of glutamate receptor channels in neurons and how it is regulated by syna ptic activity. In this study, we have investigated the phosphorylation of the AMPA-preferring glutamate receptor subunit GluR1 in cortical n eurons in primary culture. These neurons elaborate extensive processes , form functional synapses, and exhibit spontaneous 4-8 sec bursts of synaptic activity every 15-20 sec. In cultures in which this synaptic activity was suppressed by tetrodotoxin and MK-801, the GluR1 protein was phosphorylated on serine residues within a single tryptic phosphop eptide, as determined by phosphoamino acid analysis and phosphopeptide mapping. This same peptide was basally phosphorylated in recombinant GluR1 receptors transiently expressed in human embryonal kidney 293 ce lls. Treatment of these synaptically inactive cortical neurons with th e adenylyl cyclase activator forskolin resulted in a robust increase i n phosphorylation on serine residues on a phosphopeptide distinct from the basally phosphorylated peptide. Again, this same phosphopeptide w as observed in recombinant GluR1 receptors isolated from 293 cells coe xpressing the catalytic subunit of cAMP-dependent protein kinase. Spon taneous synaptic activity in cultures of cortical neurons resulted in a consistent, rapid (within 10-30 sec) increase in phosphorylation on serine and threonine residues. Interestingly, these phosphopeptides we re also phosphorylated when neurons from inactive cultures were stimul ated with phorbol esters, which activate protein kinase C. These resul ts indicate that AMPA receptors containing the GluR1 subunit may be re gulated by extracellular signals working through the cAMP second messe nger system as well as by synaptic activity, possibly acting through p rotein kinase C. Such regulation by protein phosphorylation may be inv olved in short-term changes in synaptic efficacy thought to involve th e functional modulation of AMPA receptors.