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Titolo:
Identification of substrate specificity determinants in human cAMP-specific phosphodiesterase 4A by single-point mutagenesis
Autore:
Richter, W; Unciuleac, L; Hermsdorf, T; Kronbach, T; Dettmer, D;
Indirizzi:
Univ Leipzig, Fac Med, Inst Biochem, D-04103 Leipzig, Germany Univ Leipzig Leipzig Germany D-04103 t Biochem, D-04103 Leipzig, Germany ASTA Medica, Biochem Dresden, D-01445 Radebeul, Germany ASTA Medica Radebeul Germany D-01445 Dresden, D-01445 Radebeul, Germany
Titolo Testata:
CELLULAR SIGNALLING
fascicolo: 3, volume: 13, anno: 2001,
pagine: 159 - 167
SICI:
0898-6568(200103)13:3<159:IOSSDI>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
CYCLIC-NUCLEOTIDE PHOSPHODIESTERASES; TERMINAL SPLICE REGION; CATALYTIC DOMAIN; PROTEIN-KINASE; SECONDARY-STRUCTURE; BINDING; CGMP; FAMILY; HELIX; PHOSPHORYLATION;
Keywords:
cAMP-specific phosphodiesterase 4A; substrate specificity; single-point mutagenesis; aspartic acid 440;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
43
Recensione:
Indirizzi per estratti:
Indirizzo: Dettmer, D Univ Leipzig, Fac Med, Inst Biochem, Liebigstr 16, D-04103 Leipzig, Germany Univ Leipzig Liebigstr 16 Leipzig Germany D-04103 zig, Germany
Citazione:
W. Richter et al., "Identification of substrate specificity determinants in human cAMP-specific phosphodiesterase 4A by single-point mutagenesis", CELL SIGNAL, 13(3), 2001, pp. 159-167

Abstract

To identify amino acids that might be involved in discriminating guanosine-3',5'-cyclic phosphate (cGMP) towards adenosine-3',5'-cyclic phosphate (cAMP) binding in the cAMP-specific phosphodiesterases, alignments of different human cyclic nucleotide phosphodiesterases (PDEs) were performed. Eight amino acid residues that are highly conserved in the cAMP-hydrolysing phosphodiesterases (PDE1, PDE3, PDE4, PDE7, PDE8) and that did not show any homologies to the cGMP-specific phosphodiesterases (PDE5, PDE6, PDE9) were selected 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 HSPDE4A4B; accession number L20965) were generated and expressed in COS1 cells. The expression products were characterised with regard to cAMP and cGMP hydrolysis and sensitivity towards type-specific inhibitors. The mutation of Phe484 toward Tyr, Ala590 toward Cys, Leu391 and Val501 towards Ala had no significant influence on substrate affinity or specificity. However, the exchange of Trp375 and Trp605 for aliphatic residues abolished catalytic activity and the exchange of Pro595 for Ile led to sevenfold decrease of substrate affinity and an 14-fold decrease of the affinity towards the PDE4-specific 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 substrate specificity was altered from the highly specific cAMP hydrolysis to an equally efficient cAMP and cGMP binding and hydrolysis. In addition, the IC50 values for common PDE4-specific inhibitors like rolipram, N-(3,5-dichlorpyrid-4-yl)-3-cyclopentyl-oxy-4-methoxy-benzamide (RPR-73401) and 8-methoxy-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 residue suggests that Asp440 may play a similar role in other cAMP-PDEs. (C) 2001 Elsevier Science Inc. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 15/01/21 alle ore 22:49:00