Identification of substrate specificity determinants in human cAMP-specific phosphodiesterase 4A by single-point mutagenesis

To identify amino acids that might be involved in discriminating guanosine- 3',5'-cyclic phosphate (cGMP) towards adenosine-3',5'-cyclic phosphate (cAM P) binding in the cAMP-specific phosphodiesterases, alignments of different human cyclic nucleotide phosphodiesterases (PDEs) were performed. Eight am ino acid residues that are highly conserved in the cAMP-hydrolysing phospho diesterases (PDE1, PDE3, PDE4, PDE7, PDE8) and that did not show any homolo gies to the cGMP-specific phosphodiesterases (PDE5, PDE6, PDE9) were select ed from these alignments. Using the technique of site-directed mutagenesis, derivatives of PDE4A carrying single mutations at these conserved residues (amino acid positions are given according to the human PDE4A isoform HSPDE 4A4B; accession number L20965) were generated and expressed in COS1 cells. The expression products were characterised with regard to cAMP and cGMP hyd rolysis and sensitivity towards type-specific inhibitors. The mutation of P he484 toward Tyr, Ala590 toward Cys, Leu391 and Val501 towards Ala had no s ignificant influence on substrate affinity or specificity. However, the exc hange of Trp375 and Trp605 for aliphatic residues abolished catalytic activ ity and the exchange of Pro595 for Ile led to sevenfold decrease of substra te affinity and an 14-fold decrease of the affinity towards the PDE4-specif ic inhibitor 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) . Both effects may provide evidence for a structural importance of Trp375, Trp605 and Pro595 for PDE function. By exchanging the aspartate residue for asparagine or alanine at position 440 of the human PDE4A4B isoform, the su bstrate specificity was altered from the highly specific cAMP hydrolysis to an equally efficient cAMP and cGMP binding and hydrolysis. In addition, th e IC50 values for common PDE4-specific inhibitors like rolipram, N-(3,5-dic hlorpyrid-4-yl)-3-cyclopentyl-oxy-4-methoxy-benzamide (RPR-73401) and 8-met hoxy-5-N-propyl-3-methyl-1-ethyl-imidazo[1,5-a]-pyrido[3,2-e]-pyrazinone (D -22888) were dramatically increased. These results demonstrate an important role of the aspartate at position 440 in determining substrate specificity and inhibitor susceptibility of PDE4A. The strong conservation of this res idue suggests that Asp440 may play a similar role in other cAMP-PDEs. (C) 2 001 Elsevier Science Inc. All rights reserved.