Abstract
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.
Highlights
PDEs1 are expressed as a family of distinct isoforms with each subgroup of isoforms containing multiple spliced variants [1]
PDE-6g Inhibits protein kinase A (PKA)-activated PDE-5 Activity—To explore the hypothesis that PDE-5 can be regulated by small proteins homologous to PDE-6g, we have tested the ability of recombinant PDE-6g to modulate the activity of the partially purified PDE-5
Activation of Membrane-bound PDE-5 by PKA—The finding that a peptide corresponding to amino acids 24 – 46 of PDE-6g can modulate the activation of PDE-5 and that an antibody raised to this region identified two low molecular mass proteins suggests that these proteins may function like PDE-6g
Summary
All biochemicals were from Boehringer Mannheim, whereas general chemicals were from Sigma. [g-32P]ATP, [3H]cGMP, and excitation chemiluminescence detection kits were from Amersham International (Bucks, UK). Purified brain protein kinase C containing multiple isoforms was purchased from Calbiochem
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