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Comparative Study
. 1999 Jun;10(6):1811-20.
doi: 10.1091/mbc.10.6.1811.

A constitutively "phosphorylated" guanylyl cyclase-linked atrial natriuretic peptide receptor mutant is resistant to desensitization

L R Potter  1 , T Hunter
Affiliations
Free PMC article
Comparative Study

A constitutively "phosphorylated" guanylyl cyclase-linked atrial natriuretic peptide receptor mutant is resistant to desensitization

L R Potter et al. Mol Biol Cell. 1999 Jun.
Free PMC article

Abstract

Dephosphorylation of the natriuretic peptide receptor-A (NPR-A) is hypothesized to mediate its desensitization in response to atrial natriuretic peptide (ANP) binding. Recently, we identified six phosphorylation sites within the kinase homology domain of NPR-A and determined that the conversion of these residues to alanine abolished the ability of the receptor to be phosphorylated or to be activated by ANP and ATP. In an attempt to generate a form of NPR-A that mimics a fully phosphorylated receptor but that is resistant to dephosphorylation, we engineered a receptor variant (NPR-A-6E) containing glutamate substitutions at all six phosphorylation sites. Consistent with the known ability of negatively charged glutamate residues to substitute functionally, in some cases, for phosphorylated residues, we found that NPR-A-6E was activated 10-fold by ANP and ATP. As determined by guanylyl cyclase assays, the hormone-stimulated activity of the wild-type receptor declined over time in membrane preparations in vitro, and this loss was blocked by the serine/threonine protein phosphatase inhibitor microcystin. In contrast, the activity of NPR-A-6E was more linear with time and was unaffected by microcystin. The nonhydrolyzable ATP analogue adenosine 5'-(beta,gamma-imino)-triphosphate was half as effective as ATP in stimulating the wild-type receptor but was equally as potent in stimulating NPR-A-6E, suggesting that ATP is required to keep the wild-type but not 6E variant phosphorylated. Finally, the desensitization of NPR-A-6E in whole cells was markedly blunted compared with that of the wild-type receptor, consistent with its inability to shed the negative charge from its kinase homology domain via dephosphorylation. These data provide the first direct test of the requirement for dephosphorylation in guanylyl cyclase desensitization and they indicate that it is an essential component of this process.

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Figures

Figure 1
Figure 1
NPR-A undergoes agonist-dependent (homologous) desensitization. (A) NPR-A desensitization is biphasic. 293 cells stably expressing NPR-A were treated with 1 μM ANP for increasing periods, and then crude membranes were prepared and assayed for guanylyl cyclase activity in the presence of the activators indicated or fractionated by SDS-PAGE, blotted to Immobilon membrane, and probed with a NPR-A–specific antiserum. The antigen–antibody complex was quantitated by incubating with an 125I-coupled donkey anti-rabbit–directed secondary antibody. Control equals no ANP incubation. (B) Long-term declines in ANP-dependent activity are explained by decreases in NPR-A protein. The ratio of ANP/ATP/Mg2+-dependent over Mn2+ and Triton X-100-dependent guanylyl cyclase activities from A (ordinate) was plotted versus time of ANP incubation (abscissa). The vertical bars contained within each symbol represent the range of values obtained from two separated samples, which were determined in duplicate. For some points the range is smaller than the diameter of the symbol and is not visible.
Figure 2
Figure 2
Description of wild-type and phosphorylation site mutant NPR-A receptors. LBD, ligand-binding domain; TM, transmembrane domain; GCD, guanylyl cyclase domain.
Figure 3
Figure 3
Glutamate but not alanine phosphorylation site substitutions yield hormonally responsive receptors. (A) Glutamate-substituted receptors are activated by ANP and ATP. Human 293 cells were transiently transfected with the indicated NPR-A expression constructs; 48 h later, crude membranes were prepared and assayed for guanylyl cyclase activity in the presence of Mg2+-GTP only (basal) or ANP, ATP, and Mg2+-GTP (ANP/ATP). (B) The hormone-dependent activities of the glutamate-substituted receptors are not explained by increased protein levels. Crude membranes were prepared as described above and assayed for guanylyl cyclase activity in the presence of Triton X-100 and Mn2+-GTP. (C) The glutamate substitutions are not equivalent to the wild-type receptor. The activity ratio of the wild-type and mutant receptors was determined by dividing the hormone-dependent activity by the detergent activity and multiplying this number by 100. The vertical bars centered above the columns represent the range of values obtained from four separate determinations, which were determined in two separate experiments.
Figure 4
Figure 4
ATP and AMPPNP equivalently activate NPR-A-6E but not the wild-type receptor. Human 293 cells were transiently transfected with either wild-type NPR-A or NPR-A-6E expression constructs; 48 h later, crude membranes were prepared, and guanylyl cyclase activities were determined in the presence of 1 μM ANP and 1 mM adenine nucleotide (AMPPNP or ATP) and in the presence or absence of 1 μM microcystin (MC) for 5 min at 37°C. The vertical bars centered above the columns represent the range of values obtained from two separate determinations, which were determined in duplicate.
Figure 5
Figure 5
Microcystin blocks the decline in the activity of wild-type NPR-A but not NPR-A-6E in vitro. Human 293 cells were transiently transfected with either the NPR-A (W.T., squares) or NPR-A-6E (6E, circles) expression constructs; 48 h later, crude membranes were prepared, and hormone-dependent guanylyl cyclase activity (ANP, ATP, and Mg2+-GTP) was determined for the indicated periods in the presence (filled symbols) or absence (open symbols) of 1 μM microcystin.
Figure 6
Figure 6
NPR-A-6E displays a blunted desensitization response in whole cells. (A) Effect of ANP incubation on subsequent ANP- and detergent-dependent guanylyl cyclase activities. Human 293 cells were transiently transfected with either NPR-A (W.T.) or NPR-A-6E (6E) expression constructs. Two days later, the cells were incubated with 0.5 μM ANP for the indicated periods. The cells were then washed twice with PBS, and crude membranes were prepared and assayed for guanylyl cyclase activity in the presence of ANP, ATP, and Mg2+-GTP or Triton X-100 and Mn2+-GTP. (B) Effect of ANP incubation on the activation ratio of NPR-A-6E. The ratios of hormone-dependent over detergent-dependent guanylyl cyclase activities (ordinate) were plotted versus time of ANP incubation (abscissa). The vertical bars contained within each symbol represent the range of two separate determinations.
Figure 7
Figure 7
Cells expressing NPR-A-6E but not wild-type NPR-A demonstrate linear ANP-dependent cGMP accumulation. Human 293 cells were transiently transfected with either NPR-A (W.T.) or NPR-A-6E (6E) expression constructs and then split into 12-well plates 24 h later. The next day these cells were moved to ambient temperature and incubated with 0.5 ml of DMEM containing 0.5 mM 1-methyl-3-isobutylxanthine and 200 μM ANP for the periods shown. cGMP production was terminated by adding 0.5 ml of 6% trichloroacetic acid to each well at the indicated time and assayed as described in MATERIALS AND METHODS. The vertical bars contained within each symbol represent the SEM, where n = 4.
Figure 8
Figure 8
Mutation of Ser-497 or Thr-500 does not block the desensitization of NPR-A. Human 293 cells were transiently transfected with the indicated NPR-A expression constructs; 48 h later the cells were washed and then incubated in the presence (Desensitized) or absence (Control) of 200 μM ANP for 1 h. Crude membranes were prepared and assayed for guanylyl cyclase activity in the presence of ANP, ATP, and Mg2+-GTP (A) or Mn2+-GTP and 1% Triton X-100 (B). The vertical bars centered above the columns represent the range of values obtained from two separate determinations, which were determined in duplicate. When the error is <3%, the error bars are not shown.
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References

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