IDENTIFICATION, CHARACTERIZATION AND REGIONAL DISTRIBUTION IN BRAIN OF RPDE-6 (RNPDE4A5), A NOVEL SPLICE VARIANT OF THE PDE4A CYCLIC-AMP PHOSPHODIESTERASE FAMILY

COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE 4AS). This led to the expression of a novel, cyclic AMP-specific, roli pram-inhibited phosphodiesterase activity. In such transfected cells a novel similar to 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant, RD1 (RNPDE 4A1A). RPDE-6 activity and immunoreactivity was found distributed betw een both pellet (similar to 25%) and cytosol (similar to 75%) fraction s of transfected COS-7 cells. Soluble and pellet RPDE-B activities exh ibited similar low K-m values for cyclic AMP (similar to 2.4 mu M) and were both inhibited by low concentrations of rolipram, with IC50 valu es for the soluble activity being lower (similar to 0.16 mu M) than fo r the pellet activity (similar to 1.2 mu M). Pellet RPDE-6 was resista nt to release by either high NaCl concentrations or the detergent Trit on X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an similar to 79 k Da species reflecting RD1 and an similar to 109 kDa species that co-mi grated with the immunoreactive species seen in COS cells transfected t o express RPDE-6. The similar to 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-tr ansfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immunoprecipitated by these antisera from brain cytosol had the characteristics of COS cell-expre ssed RPDE-6 with K-m(cyclic) AMP similar to 3.7 mu M and IC(50)rolipra m similar to 0.12 mu M. The distribution of PDE activity immunoprecipi tated from the cytosol of various brain regions paralleled that seen f or the distribution of the similar to 109 kDa immunoreactive species. It is suggested that the 109 kDa species identified in brain cytosol a nd pellet fractions is the native form of RPDE-6. The PDE4A splice var iants, RD1 and RPDE-6, were shown to have distinct patterns of express ion among various brain regions. PDE4A and PDE4B activities appear to provide the major source of PDE4 activity in brain membranes, whereas the cytosolic PDE4 activity is suggested to reflect predominantly the activity of the PDE4D family. Alternative splicing of the PDE4A gene c onfers distinct N-terminal domains on RPDE-6 and RD1, which attenuates the V-max, of these enzymes and defines their distinct subcellular di stribution pattern.