Abstract
Our previous studies have suggested that two phosphodiesterase type 10A (PDE10A) variants, PDE10A1 and PDE10A2 transcripts, are mainly expressed in humans and that PDE10A2 and PDE10A3 transcripts are major variants in rats. In the present study, immunoblot analysis demonstrated that PDE10A proteins, especially PDE10A2, are more abundant in membrane fractions than in cytosolic fractions of rat striatum. Recombinant PDE10A1 and PDE10A3 were produced only in cytosolic fractions of transfected PC12h cells. By contrast, recombinant PDE10A2 was present mainly in membrane fractions. This finding agreed well with the result of subcellular fractionation of PDE10A in rat striatum. Immunocytochemical analysis showed that PDE10A2 was localized in the Golgi apparatus of transfected PC12h cells. PDE10A2 was phosphorylated by cAMP-dependent protein kinase (PKA) at Thr16. Interestingly, recombinant protein of wild-type PDE10A2, but not PDE10A2 mutant with an Ala replacement at Thr16, was distributed to cytosolic fractions by co-transfection with a plasmid encoding the catalytic subunit of PKA. A PDE10A2 mutant with Glu substitution at Thr16, which can be a mimic of phosphorylation, was localized in the cytosolic fractions of transfected PC12h cells. These observations implied that phosphorylation of PDE10A2 at Thr16 by PKA caused alteration of subcellular localization of PDE10A2 from the Golgi apparatus to cytosol. It is hypothesized that cAMP signaling in the Golgi area and the cytosol in neurons is controlled through alteration of subcellular localization of PDE10A brought by activation of PKA in response to intracellular elevations of cAMP.
Highlights
Cyclic nucleotides, cAMP and cGMP, control physiological functions in neuronal networks
The cellular compartmentalization of cAMP signaling through association with several forms of protein kinase A-anchoring proteins (AKAPs), which interact with the regulatory subunit of PKA, has been discussed [6]
We have previously reported that phosphodiesterase type 10A (PDE10A) transcripts and their enzymatic activities are abundant in the striatum and testis [12], and more detailed expression patterns of PDE10A transcripts and proteins in rat brain have been recently reported [13]
Summary
CAMP and cGMP, control physiological functions in neuronal networks. Proteins immunoreactive with purified anti-PDE10A antibody were observed only in fractions 16 –20 (cytosolic fractions) and fractions 14 –20 (membrane fractions), indicating that cAMP hydrolytic activities in these fractions contained PDE10A (Fig. 2, B and C).
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