The regulation of the cGMP-binding cGMP phosphodiesterase by proteins that are immunologically related to gamma subunit of the photoreceptor cGMP phosphodiesterase.
The cGMP phosphodiesterase from retinal rods (PDE-6) is an alphabetagamma2 heterotetramer. The alpha and beta subunits contain catalytic sites for cGMP hydrolysis, whereas the gamma subunits serve as a protein inhibitor of the enzyme. Visual excitation of photoreceptors enables the activated GTP-bound form of the G-protein transducin to remove the inhibitory action of the gamma subunit, thereby triggering PDE-6 activation. The type 5 phosphodiesterase (PDE-5) isoform shares a number of similar characteristics with PDE-6, including binding of cGMP to noncatalytic sites, the cyclic nucleotide specificity, and inhibitor sensitivities. Although the functional role of PDE-5 remains unclear, it has been shown to be activated by protein kinase A (PKA) (Burns, F., Rodger, I. W. & Pyne, N. J. (1992) Biochem. J. 283, 487-491). Here we report that both the recombinant gamma subunit and a peptide corresponding to amino acids 24-46 in this protein inhibited the activation of PDE-5 by PKA. Furthermore, immunoblotting airway smooth muscle membranes with a specific antibody against amino acids 24-46 of the PDE-6 gamma subunit identified two major immunoreactive small molecular mass proteins of 14 and 18 kDa (p14 and p18). These appear to form a complex with PDE-5, because PDE activity was immunoprecipitated using antibody against the PDE-6 gamma subunit. p14 and p18 were also substrates for phosphorylation by a unidentified kinase that was stimulated by a pertussis toxin-sensitive G-protein. Phosphorylation of p14/p18 in membranes treated with guanine nucleotides correlated with a concurrent reduction in the activation of PDE-5 by PKA. We suggest that p14 and p18 share an epitope common to PDE-6 gamma and that this region may interact with PDE-5 to prevent its activation by PKA.
Lochhead
A
A
Department of Physiology and Pharmacology, University of Strathclyde, Glasgow G1 1XW, Scotland.
Nekrasova
E
E
Arshavsky
V Y
VY
Pyne
N J
NJ
D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D000906:Antibodies; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D006152:Cyclic GMP / Q000378:metabolism*; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D006168:Guinea Pigs; D006706:Homeostasis; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D046911:Macromolecular Substances; D009130:Muscle, Smooth / Q000201:enzymology; D037342:Pertussis Toxin; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017948:Retinal Rod Photoreceptor Cells / Q000201:enzymology*; D014132:Trachea / Q000201:enzymology; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D000906:Antibodies; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D010566:Virulence Factors, Bordetella; D006165:Guanylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D037342:Pertussis Toxin; D017868:Cyclic AMP-Dependent Protein Kinases; D010727:Phosphoric Diester Hydrolases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D054706:Cyclic Nucleotide Phosphodiesterases, Type 5; D019204:GTP-Binding Proteins; D006152:Cyclic GMP