Engineered deletion of the unique N-terminal domain of the cyclic AMP-specific phosphodiesterase RD1 prevents plasma membrane association and the attainment of enhanced thermostability without altering its sensitivity to inhibition by rolipram

Abstract

Full-length cDNA for the rat brain rolipram-sensitive cyclic AMP phosphodiesterase (PDE), RD1 was introduced into the expression vector pSVL. COS cells transfected with the recombinant vector pSVL-RD1 exhibited a 30-55% increase in homogenate PDE activity, which was abolished by rolipram (10 microM). Removal of the first 67 nucleotides of the RD1 cDNA yielded a truncated enzyme called Met26-RD1 which lacked the N-terminal first 25 amino acids. Whereas approx. 75% of RD1 activity was membrane-associated, Met26-RD1 activity was found exclusively in the cytosol fraction. Expression of RD1 nearly doubled membrane-associated PDE activity, while expression of Met26-RD1 increased cytosolic activity by approx. 30%. Membrane RD1 activity was found to be primarily associated with the plasma membrane, was not released by either high concentrations of NaCl or by a 'hypotonic shock' treatment, but was solubilized with low concentrations of Triton X-100. Phase separation of membrane components with Triton X-114 showed partition of RD1 into both the aqueous and detergent-rich phases, whereas Met26-RD1 partitioned exclusively into the aqueous phase. Both RD1 and Met26-RD1 specifically hydrolysed cyclic AMP; were unaffected by either Ca2+/calmodulin or by low cyclic GMP concentrations; exhibited linear Lineweaver-Burke plots with similar Km values for cyclic AMP (4 microM); both were potently and similarly inhibited by rolipram (Ki approx. 0.5 microM) and were similarly inhibited by cilostamide and 3-isobutyl-1-methylxanthine. Thermal inactivation, at 50 degrees C, showed that while the cytosolic-located fraction of RD1 (t0.5 approx. 3 min) and Met26-RD1 (t0.5 approx 3 min) were similarly thermolabile, membrane-bound RD1 was considerably more thermostable (t0.5 approx. 11 min). Treatment of both cytosolic RD1 and Met26-RD1 with Triton X-100 did not affect their thermostability, but solubilization of membrane RD1 activity with Triton X-100 markedly decreased its thermostability (t0.5 approx. 5 min). The N-terminal domain of RD1 appears not to influence either the substrate specificity or inhibitor sensitivity of this enzyme, but it does contain information which can allow RD1 to become plasma membrane-associated and thereby adopt a conformation which has enhanced thermostability.

Isolation and Characterization of PDE9A, a Novel Human cGMP-specific Phosphodiesterase

“…2) in the human PDE9A putative regulatory domain (amino acids 1-287) that are absent in the splice variant cloned in mouse. This suggests that alternate splicing of the putative regulatory domain is likely to occur in PDE9A, because it has been observed in other PDE families, where it can alter membrane targeting (16) and interaction with the v-Src SH3 domain (17). The PDE9A regulatory domain is otherwise 98% identical between the species, clearly indicating that these are two members of the same PDE family and almost certainly homologues of the same isozyme.…”

Expression and regulation of human and rat phosphodiesterase type IV isogenes

“…To explain the benefit for the cell of the expression of the multiple isoforms, it needs to be discovered whether functional or regulatory differences between these isoforms exist or whether they are present in different subcellular compartments. In this respect it is interesting to note that rPDE IV-A (RD 1) was found as a membrane-bound isoform [27,28] whereas rPDE IV-D was found in soluble fractions of stimulated Sertoli cells by other authors [13,20].…”

N termini of apPDE4 isoforms are responsible for targeting the isoforms to different cellular membranes

“…Among PDE4s, the brain-specific supershort PDE4A1 is a unique membrane-associated PDE4 that binds with certain types of lipids (Shakur et al 1993). In PDE4A1, 25 amino acids (aa) of the N terminus are involved in membrane association via two different motifs, namely, helix-1 and helix-2 (Huston et al 2006).…”