PHORBOL ESTER-SENSITIVE AND GLUTAMATE-SENSITIVE PHOSPHORYLATION OF HIPPOCAMPAL MEMBRANE-PROTEINS FROM ADULT AND NEONATAL RATS

The phosphoinositide (PI) second messenger system in the neonatal rat brain is differentially stimulated as compared to that of the adult by agonists such as glutamate. Among the factors that might contribute t o the neonatal pattern is the nature of phosphorylated membrane-bound proteins which could regulate this receptor-mediated response. This st udy was undertaken to compare membrane protein phosphorylation under c onditions that affect PI hydrolysis in neonatal and adult rat hippocam pus. Two-dimensional gel analysis revealed enhanced basal phosphorylat ion of two membrane proteins (M(r): 46,000 and 80,000; pl: 4.4 and 4.2 , respectively) in the neonatal hippocampus when compared to the adult . The former phosphoprotein is present only in neonatal hippocampus. P hosphorylation of a 48,000 M(r) protein with a pI of 4.5 is prominent in hippocampal membranes from both neonatal and adult rats. After incu bation of neonatal hippocampal slices with an active phorbol ester, 12 -O-tetradecanoyl phorbol-13-acetate (TPA), all three proteins show dec reased post-hoc phosphorylation. Slices from neonatal rats incubated w ith glutamate demonstrated no alteration in the phosphorylation of any of these proteins, while those from adult rats produced a marked chan ge in phosphorylation of the 80,000 M(r) protein. The data suggest tha t phosphorylation of this protein from neonates is not yet as efficien tly coupled to receptor stimulation as that from the adult. Immunoblot analysis revealed that the 48,000 M(r) protein is the growth-associat ed protein B-50/GAP-43 and that the 80,000 M(r) protein is a membrane- associated form of the MARCKS protein. Immunoblot analysis shows that the MARCKS protein is present in the hippocampal membrane fraction and is expressed at higher levels in the neonate than in the adult. These proteins, which are differentially regulated in neonates, may mediate developmental effects of phorbol esters on PI metabolism.