A proline-rich domain in the gamma subunit of phosphodiesterase 6 mediates interaction with SH3-containing proteins (original) (raw)
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Cellular Signalling, 2012
The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g tm1 /Pde6g tm1 mice that does not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile 86 and Ile 87 residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100 µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile 86 and Ile 87 residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.
Journal of Biological Chemistry, 2010
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. . 2 The abbreviations used are: PDE6, photoreceptor cyclic nucleotide phosphodiesterase; P␣, catalytic dimer of PDE6 ␣and -subunits; P␥, inhibitory ␥ subunit of PDE6; T␣*, activated transducin ␣-subunit; GTP␥S, guanosine 5Ј-O-(thiotriphosphate).
Rod phosphodiesterase-6 PDE6A and PDE6B Subunits Are Enzymatically Equivalent
Journal of Biological Chemistry, 2010
Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K m (20-23 M) and k cat (4200-5100 s ؊1) values. Likewise, the K i values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone-and rod-specific PDE6 ␥-subunits (P␥) were comparable. Recombinant cone transducin-␣ (G␣ t2) and native rod G␣ t1 fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of G␣ t2 or G␣ t1. Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with P␥ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.
Journal of Biological Chemistry, 2011
Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (␣), while the catalytic core of cone PDE6 (␣) is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl ؊/؊ cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (␣) and the absence of cone PDE6 (␣) catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce Scone responses. However, Scone responses and light-dependent cGMP hydrolysis were eliminated when the -subunit of rod PDE6 was removed (Nrl ؊/؊ cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.
Removal of phosphorylation sites of subunit of phosphodiesterase 6 alters rod light response
The Journal of Physiology, 2006
The phosphodiesterase 6 γ (PDE6γ) inhibitory subunit of the rod PDE6 effector enzyme plays a central role in the turning on and off of the visual transduction cascade, since binding of PDE6γ to the transducin α subunit (Tα) initiates the hydrolysis of the second messenger cGMP, and PDE6γ in association with RGS9-1 and the other GAP complex proteins (Gβ5, R9AP) accelerates the conversion of TαGTP to TαGDP, the rate-limiting step in the decay of the rod light response. Several studies have shown that PDE6γ can be phosphorylated at two threonines, T22 and T35, and have proposed that phosphorylation plays some role in the physiology of the rod. We have examined this possibility by constructing mice in which T22 and/or T35 were replaced with alanines. Our results show that T35A rod responses rise and decay more slowly and are less sensitive to light than wild-type (WT). T22A responses show no significant difference in initial time course with WT but decay more rapidly, especially at dimmer intensities. When the T22A mutation is added to T35A, double mutant rods no longer showed the prolonged decay of T35A rods but remained slower than WT in initial time course. Our experiments suggest that the polycationic domain of PDE6γ containing these two phosphorylation sites can influence the rate of PDE6 activation and deactivation and raise the possibility that phosphorylation or dephosphorylation of PDE6γ could modify the time course of transduction, thereby influencing the wave form of the light response.
Experimental Eye Research, 2006
We have established earlier that rod photoreceptor cGMP-phosphodiesterase (PDE6) alpha and beta subunits are equally represented in the retina at the protein level and have similar turnover rates. mRNA quantification revealed five PDE6beta messages for every PDE6alpha transcript pointing at post-transcriptional regulation of PDE6alpha and PDE6beta expression. Indeed, the wild-type PDE6alpha mRNA was translated 5-fold more efficiently than that of PDE6beta. The coding regions of these subunits had a major contribution in this process. Here, we extend our study of translational regulation of PDE6 subunits and present a detailed analysis of the role of PDE6alpha and PDE6beta 5 0 -and 3 0 -UTRs (untranslated regions) in this process. We showed that both the short and long PDE6beta 5 0 -UTRs lead to more efficient protein synthesis than the PDE6alpha 5 0 -UTR. The 3 0 -UTRs of PDE6alpha and PDE6beta stimulated translation by approximately 2-and 3-fold, respectively. However, the positive effect of the PDE6alpha or PDE6beta 3 0 -UTRs was not observed when these regions were placed in constructs containing the 5 0 -UTR of the corresponding PDE6 subunit. Furthermore, it appears that PDE6alpha 5 0 -and 3 0 -UTRs may be involved in a base pairing interaction that reduces the efficiency of protein synthesis. Finally, using progressive deletion analysis of the PDE6alpha 5 0 -UTR, we have identified several regions that have significant contribution in regulation of protein synthesis. Based on these and earlier published data, it can be stated that an equimolar level of PDE6alpha and PDE6beta synthesized from different amounts of mRNA (ratio of PDE6alpha to PDE6beta mRNA in the retina is 1:5) is achieved as a result of combinatorial effects of 5 0 -UTRs and coding regions of PDE6alpha and PDE6beta mRNAs on translational regulation.
Prenylation is the posttranslational modification of a carboxylterminal cysteine residue of proteins that terminate with a CAAX motif. Following prenylation, the last three amino acids are cleaved off by the endoprotease, RAS-converting enzyme 1 (RCE1), and the prenylcysteine residue is methylated. Although it is clear that prenylation increases membrane affinity of CAAX proteins, less is known about the importance of the postprenylation processing steps. RCE1 function has been studied in a variety of tissues but not in neuronal cells. To approach this issue, we generated mice lacking Rce1 in the retina. Retinal development proceeded normally in the absence of Rce1, but photoreceptor cells failed to respond to light and subsequently degenerated in a rapid fashion. In contrast, the inner nuclear and ganglion cell layers were unaffected. We found that the multimeric rod phosphodiesterase 6 (PDE6), a prenylated protein and RCE1 substrate, was unable to be transported to the outer segments in Rce1-deficient photoreceptor cells. PDE6 present in the inner segment of Rce1-deficient photoreceptor cells was assembled and functional. Synthesis and transport of transducin, and rhodopsin kinase 1 (GRK1), also prenylated substrates of RCE1, was unaffected by Rce1 deficiency. We conclude that RCE1 is essential for the intracellular trafficking of PDE6 and survival of photoreceptor cells.
Biochemical and Biophysical Research Communications, 2009
The γ subunit of rod-specific cGMP phosphodiesterase 6 (PDE6γ), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6γ on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6γ may increase or decrease the ability of PDE6γ to deactivate phototransduction. To resolve role of phosphorylation of PDE6γ in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6γ (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6γ, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6γ is essential for the regulation of G-protein signaling.
Genomics, 1998
rods and two a in cones) and two copies of inhibitory The mammalian multisubunit photoreceptor cGMP small subunits (rod g and cone g) (Baehr et al., 1979; phosphodiesterase PDEabg 2 (PDE6 family) is a pe- Hurwitz et al., 1985; Deterre et al., 1988; Gillespie and ripherally membrane-associated enzyme. A novel sub- . Under physiological unit, termed PDEd (HGMW-approved symbol, PDE6D; conditions, most of PDE is peripherally bound to rod MW 17 kDa), is able to detach PDE partially from boor cone outer segment membranes via methylated and vine rod outer segment membranes under physiologiisoprenylated C-termini of the large subunits (Swanson cal conditions. Cloning of human and mouse PDEd and Applebury, 1984; cDNAs revealed that PDEd is a nearly perfectly con-1992; . A 17-kDa served polypeptide of 150 amino acids that shows parpolypeptide, referred to as PDEd subunit, copurified tial sequence homology to photoreceptor RG4 of unwith a soluble fraction of bovine rod and cone PDE and known function. Multiple-species Southern blot analywas cloned recently Florio et al., sis demonstrates that the PDEd gene has been well 1996). PDEd does not affect the PDE catalytic activity conserved during evolution and is detectable at high in vitro, but is thought to directly interact with isoprenstringency in invertebrates. The human and mouse ylated C-termini, preventing membrane anchoring. genes are contained in less than 8 kb of genomic DNA The physiological significance of partial PDE solubiliand consist of four exons and three introns (0.7-4 kb zation is unclear, but PDE detachment may serve as a in human, 0.7-2.2 kb in mouse). The PDEd gene strucmechanism to desensitize the phototransduction casture is identical to that of the C27H5.1 gene identified cade by removing active PDE from the membrane surin the eyeless nematode Caenorhabditis elegans. The face ( Florio et al., 1996).