The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its ␥-subunit (P␥), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of P␥ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P␥78 -87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P␥ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P␥ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact ␣2 helix of P␥ stabilize binding of C-terminal P␥ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P␥ molecule and tested for activation by transducin, we found that the C-terminal region (P␥63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P␥ truncation mutants. Our results are consistent with two distinct mechanisms of P␥ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of P␥ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of P␥ to effectively relieve its inhibitory constraint. The photoreceptor cyclic nucleotide phosphodiesterase (PDE6)2 is the central enzyme in the vertebrate visual signaling pathway in rods and cones. Phototransduction is initiated when light induces the isomerization of the 11-cis-retinal chromophore of rhodopsin, which leads to activation of the photoreceptor-specific G-protein, transducin. Transducin binds GTP and releases its activated ␣-subunit (T␣*-GTP) to activate membrane-associated rod PDE6 by relieving the inhibition of the ␥-subunit at the active sites. The activation of PDE6 results in rapid lowering of cGMP levels, closure of cGMP gated ion channels, and hyperpolarization of the cell membrane (1-5).The PDE6 holoenzyme consists of a catalytic dimer of ␣-and ␤-subunits (P␣␤) and two inhibitory ␥-subunits (P␥) that are tightly bound to P␣␤. Considering its small size, the P␥ subunit of rod and cone PDE6 serves a remarkable number of functions (reviewed in Refs. (6 and 7): 1) a primary function of the P␥ subunit is to inhibit catalysis of cGMP by binding to the catalytic domain of PDE6; 2) the P␥ subunit also enhances the binding affinity of cGMP to the regulatory GAF (cGMP-regulated PDEs, certain adenylate cyclases, and the transcription factor Fh1A of bacteria) domain; 3) activated transducin binds to the P␥ subunit, leading to deinhibition of PDE6 at its active site; and 4) during deactivation, the ␥-subunit participates in a protein complex with the RGS9 and o...

IL-23 is a novel cytokine composed of an IL-12 p40 and a p19 subunit. We analyzed human peripheral blood mononuclear cells (PBMC) and hematopoietic cell lines for constitutive expression of IL-23 and IL-12 subunits and expression following exposure to various stimuli, and investigated the mechanisms of their induction by LPS and SAC. IL-23 p19 and IL-12 p35 mRNAs were expressed in fresh PBMC, and expression of all IL-23 and IL-12 subunits was up-regulated by LPS and SAC. LPS-induced increase of IL-23 and IL-12 subunits expression was CD14-dependent, while CD14 was not involved in SAC-induced p19 transcription. Both LPS- and SAC-induced subunits expression required p38 MAPK pathway. PHA effected an increase of p19 mRNA in both CD4+ and CD8+ T cells, whereas p35 was minimally regulated by PHA. IFN-gamma primed monocytes for LPS stimulation of p19, p35 and p40 expression. p19 mRNA was detectable in most hematopoietic cell lines tested but p35 distribution was more restricted. A phorbol diester enhanced p19, p35 and p40 expression in EBV-positive cell lines. Our results suggest that both the subunits of IL-23 are tightly regulated; the expression pattern and regulation mode of IL-23 p19 is similar to as well as distinct from that of IL-12 p35.