Function of calmodulin in postsynaptic densities. I. Presence of a calmodulin-activatable cyclic nucleotide phosphodiesterase activity.

Abstract

The postsynaptic density (PSD) fraction from canine cerebral cortex was found to contain an endogenous cyclic nucleotide-phosphodiesterase activity that was dependent on Mn 2+ and/or Mg" but not on Ca t + . Maximal activity was obtained at 1 gm Mn" . This cyclic nucleotide phosphodiesterase activity was not decreased upon removal of the calmodulin from the PSD fraction, nor was it increased by the addition of calmodulin to a postsynaptic density fraction deficient in calmodulin . The enzymatic activity could be extracted by sonication, with the soluble enzyme having properties similar to those found in the native structure. Two peaks of cyclic nucleotide phosphodiesterase activities could be obtained after S-300 Sephacryl column chromatography of this soluble fraction : fraction I (excluded peak) and fraction II (215,000 mol wt) . The fraction I activity preferred cyclic AMP over cyclic GMP and was not activated by calmodulin . The fraction II activity had an approximately fourfold lower K,n for cyclic GMP over cyclic AMP. This fraction II activity was activatable by calmodulin, which increased the Vmsx and decreased the Km in the case of both cyclic nucleotides. We conclude that two activities are present in the PSD, one activatable, and one not activatable, by Calmodulin .The postsynaptic density (PSD) is a prominent structure, of unknown function, in central nervous system synapses (1). As observed by electron microscopy, the PSD appears as a densely staining thickening which lies along the cytoplasmic side of the postsynaptic membrane (1) . Recently, we have isolated from canine cerebral cortex a PSD fraction that has been found to contain some 10 major and at least 20 minor proteins (10). Of the enzymatic activities tested, there are no Mg" or Cat +-ATPase (10) nor adenylate cyclase (18) activities, and very small amounts of cytochrome c oxidase (10) or 5'-nucleotidase (10) activities. However, two protein kinase activities are present, one activatable by cyclic AMP (4, 45) and one activatable by Ca" and calmodulin (7,18,20,21) ; the protein substrates, proteins Ia and Ib, which are phosphorylated by the former kinase (4, 45) and the protein substrates, the major 51,000 Mr protein and a 62,000 M, protein, which are phosphorylated by the latter kinase (7,18,20,21) are also found in the PSD preparation .Immunochemistry has been used to confirm the presence of proteins Ia and Ib in the PSD (6) . Other proteins identified to be intrinsic constituents are actin (5, 27), the alpha and beta subunits of tubulin (5,27,35,36,48) and a major M, = 51,000

Histochemistry of nucleotidyl cyclases and cyclic nucleotide phosphodiesterases

“…The building of cyclic nucleotides is influenced by several hormones or neurotransmitters and the degradation of cyclic nucleotides can also be regulated by such substances (Loten et al, 1978;Saeed et al, 1981;Nemoz & Prigent, 1984;Keppens & de Wulf, 1984;Vallet-Strouve et al, 1984;Conti et al, 1984;Teo et al, 1987;Saltiel & Steigerwalt, 1986;Nagasaka et al, 1986;Francis & Kona, 1982). Further possibilities for the regulation of cyclic nucleotide phosphodiesterases are by the regulation of cyclic nucleotides themselves (Marchmont & Houslay, 1980;Martins et al, 1982;Palmer & Doukas, 1984;Tremblay et al, 1985), regulation by nucleotide binding proteins (De Mazancourt & Giudicelli, 1984), regulation of cyclic nucleotide phosphodiesterase by proteolysis (Sakai et al, 1977(Sakai et al, , 1978Epstein et al, 1978;Kincaid et al, 1985) and regulation by calcium/calmodulin (Thompson & Appleman, 1971;Ho et al, 1976;Sakai et al, 1977;Sharma & Wang, 1986;Grab et al, 1981;Erneux et al, 1985). Four major phosphodiesterase isoenzymes have been described (Hidaka et al, 1977;Manganiello et al, 1984;Reeves et al, 1987;Vaughan et al, 1980) which may exist in further multiple forms (Minneman, 1976;Pichard & Cheung, 1976;Hidaka et al, 1984;Erneux et al, 1985;Weishaar et al, 1985).…”

“…The relationship of these isoenzymes differs considerably from one brain area to another (Greenberg et al, 1978). The isoenzyme that can be activated by calmodulin is also localized in the postsynaptic densities (Grab et al, 1981), and has a different behaviour to the two cyclic nucleotides.…”

cAMP-Dependent Protein Kinase Mediates Activity-Regulated Synaptic Targeting of NMDA Receptors

“…Decreased calcium entry through NMDA receptors could potentially activate PKA through decreased activity of a calcium/calmodulin-activated phosphodiesterase of the PDE1 family (Zhao et al, 1997;Kakkar et al, 1999). These enzymes are highly expressed in neurons, including hippocampal pyramidal neurons, are present in the postsynaptic density (Grab et al, 1981), and are targets of the inhibitor IBMX used in this study (Fig. 6).…”

Biochemical and immunochemical evidence that the "major postsynaptic density protein" is a subunit of a calmodulin-dependent protein kinase.

“…It has been proposed that they are primarily structural, serving to anchor membrane or cytosolic proteins in the region of the postsynaptic membrane (21). The fraction is enriched in cyclic nucleotide phosphodiesterase activity (22,23), calmodulin (24), and both-cAMP- (25)(26)(27) and calmodulin-dependent protein kinase activities (27)(28)(29). Immunocytochemical evidence suggests that a calmodulin-dependent protein phosphatase, calcineurin, may also be located in PSDs in situ (30,31).…”