A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Bereta, Grzegorz; Wang, Benlian; Kiser, Philip D.; Baehr, Wolfgang; Jang, Geeng Fu; Palczewski, Krzysztof

doi:10.1074/jbc.m109.061713

Cited by 22 publications

(20 citation statements)

References 55 publications

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“…Recent studies by Bereta et al, have demonstrated phosphorylation at four serine residues within the kinase homology domain of guanylyl cyclase 1 (GC1) (also known as GC-E) from mouse retina using mass spectrometry (26). Interestingly, the four phosphorylation sites identified within the kinase homology domain of GC1 align with the highly phosphorylated region of the kinase homology domains of GC-A and GC-B.…”

Section: Discussionmentioning

confidence: 99%

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Yoder¹,

Stone

Griffin

et al. 2010

Biochemistry

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Guanylyl cyclase A and B (GC-A and GC-B) are transmembrane guanylyl cyclase receptors that mediate the physiologic effects of natriuretic peptides. Some sites of phosphorylation are known for rat GC-A and GC-B, but no phosphorylation site information is available for the human homologs. Here, we used mass spectrometry to identify phosphorylation sites in GC-A and GC-B from both species. Tryptic digests of receptors purified from HEK293 cells were separated and analyzed by nLC-MS-MS. Seven sites of phosphorylation were identified in rat GC-A (S497, T500, S502, S506, S510, T513, and S487) and all of these sites except S510 and T513 were observed in human GC-A. Six phosphorylation sites were identified in rat GC-B (S513, T516, S518, S523, S526, and T529) and all six sites were also identified in human GC-B. Five sites are identical between GC-A and GC-B. S487 in GC-A and T529 in GC-B are novel, uncharacterized sites. Substitution of alanine for S487 did not affect initial ligand-dependent GC-A activity, but a glutamate substitution reduced activity 20%. Similar levels of ANP-dependent desensitization were observed for the wild type, S487A and S487E forms of GC-A. Substitution of glutamate or alanine for T529 increased or decreased ligand-dependent cyclase activity of GC-B, respectively, and T529E increased cyclase activity in a GC-B mutant containing glutamates for all five previously identified sites as well. In conclusion, we identified and characterized new phosphorylation sites in GC-A and GC-B and provide the first evidence of phosphorylation sites within human guanylyl cyclases.

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Section: Discussionmentioning

confidence: 99%

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Yoder¹,

Stone

Griffin

et al. 2010

Biochemistry

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“…42 Purification of ABCA4 was performed by immunoprecipitation on ice under dim red light. ROS from 10 mouse retinas were solubilized with 1 mL of buffer containing 10 mM sodium phosphate (pH 7.6), 50 mM NaCl, 50 mM NaF, 10 mM EDTA, 30 mM DDM, 10% glycerol, 0.1 mM DTT, and 10 nM okadaic acid.…”

Section: Methodsmentioning

confidence: 99%

Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4

et al. 2011

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ABCA4 is a photoreceptor-specific ATP-binding cassette transporter implicated in the clearance of all-trans-retinal produced in the retina during light perception. Multiple mutations in this protein have been linked to Stargardt disease and other visual disorders. Here we report the first systematic study of posttranslational modifications in native ABCA4 purified from bovine rod outer segments. Seven N-glycosylation sites were detected in exocytoplasmic domains 1 and 2 by mass spectrometry, confirming the topological model of ABCA4 proposed previously. The modifying oligosaccharides were relatively short and homogeneous, predominantly representing a high-mannose type of N-glycosylation. Five phosphorylation sites were detected in cytoplasmic domain 1, with four of them located in the linker “regulatory-like” region conserved among ABCA subfamily members. Contrary to published results, phosphorylation of ABCA4 was found to be independent of light. Using human ABCA4 mutants heterologously expressed in mammalian cells, we showed that the Stargardt disease-associated alanine mutation in the phosphorylation site at position 901 led to protein misfolding and degradation. Furthermore, replacing the S1317 phosphorylation site reduced the basal ATPase activity of ABCA4, whereas an alanine mutation in either the S1185 or T1313 phosphorylation site resulted in a significant decrease in the all-trans-retinal-stimulated ATPase activity without affecting the basal activity, protein expression, or localization. In agreement with this observation, partial dephosphorylation of native bovine ABCA4 led to reduction of both basal and stimulated ATPase activity. Thus, we present the first evidence that phosphorylation of ABCA4 can regulate its function.

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“…Although the function of the KHD is unclear, it was shown to be essential for interaction with guanylate cyclase activating proteins (GCAPs) (Duda et al, 1996; Laura and Hurley, 1998). Further, it was shown that photoreceptor GC1 auto-phosphorylates serine residues of the 529-GSRSSLGARS motif (Bereta et al, 2010). The predominantly outer segment localization of GCct4 suggested the possibility that the KHD might contain targeting information.…”

Section: Resultsmentioning

confidence: 99%

Targeting of mouse guanylate cyclase 1 (Gucy2e) to Xenopus laevis rod outer segments

et al. 2011

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Photoreceptor guanylate cyclase (GC1) is a transmembrane protein and responsible for synthesis of cGMP, the secondary messenger of phototransduction. It consists of an extracellular domain, a single transmembrane domain, and an intracellular domain. It is unknown how GC1 targets to the outer segments where it resides. To identify a putative GC1 targeting signal, we generated a series of peripheral membrane and transmembrane constructs encoding extracellular and intracellular mouse GC1 fragments fused to eGFP. The constructs were expressed in X. laevis rod photoreceptors under the control of the rhodopsin promoter. We examined the localization of GFP-GC1 fusion proteins containing the complete GC1 sequence, or partial GC1 sequences, which were membrane-associated via either the GC1 transmembrane domain or the rhodopsin C-terminal palmitoyl chains. Full-length GFP-GC1 targeted to the rod outer segment disk rims. As a group, fusion proteins containing the entire cytoplasmic domain of GC1 targeted to the OS, whereas other fusion proteins containing portions of the cytoplasmic or the extracellular domains did not. We conclude that GC1 likely has no single linear peptide-based OS targeting signal. Our results suggest targeting is due to either multiple weak signals in the cytoplasmic domain of GC1, or co-transport to the OS with an accessory protein.

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A Functional Kinase Homology Domain Is Essential for the Activity of Photoreceptor Guanylate Cyclase 1

Cited by 22 publications

References 55 publications

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Mass Spectrometric Identification of Phosphorylation Sites in Guanylyl Cyclase A and B

Posttranslational Modifications of the Photoreceptor-Specific ABC Transporter ABCA4

Targeting of mouse guanylate cyclase 1 (Gucy2e) to Xenopus laevis rod outer segments

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