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Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Galphas
1Department of Pharmacology, Mount Sinai School of Medicine, New York, NY 10029, USA.
Abstract:
Receptors activate adenylyl cyclases through the Galphas subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Galphas. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Galphas. Similar treatment of AC1 or AC2 did not affect Galphas stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Galphas stimulation. A peptide encoding the region 660-682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Galphas. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660-682 of AC6 is involved in regulation of signal transfer from Galphas. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.
Insights
Protein kinase A (PKA) selectively reduces adenylyl cyclase 6 (AC6) stimulation by G-alpha-s. Phosphorylation at Ser-674 in AC6 is key to this desensitization, revealing isoform-specific regulation of adenylyl cyclase signaling.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Receptor activation of adenylyl cyclases involves the G-alpha-s subunit.
- Heterologous desensitization in cells expressing adenylyl cyclase 6 (AC6) reduces G-alpha-s stimulation.
- Protein kinase A (PKA) plays a role in adenylyl cyclase regulation.
Purpose of the Study:
- To investigate the specific effects of PKA on adenylyl cyclase isoforms, particularly AC6.
- To identify the molecular mechanisms underlying PKA-mediated desensitization of AC6.
- To explore how isoform-specific regulation is achieved within conserved signaling pathways.
Main Methods:
- Treatment of recombinant AC6, AC1, and AC2 with PKA in insect cell membranes.
- Site-directed mutagenesis of AC6, converting Ser-674 to Alanine (Ala).
- Peptide inhibition assays using a region encompassing AC6 residues 660-682, with and without Ser-674 to Asp (Asp) substitution.
Main Results:
- PKA treatment selectively reduced G-alpha-s stimulation of AC6 at high concentrations (>10 nM), but not AC1 or AC2.
- Conversion of Ser-674 to Ala in AC6 abolished PKA phosphorylation and the subsequent loss of G-alpha-s stimulation.
- A peptide from AC6 residues 660-682 inhibited G-alpha-s stimulation of AC6 and AC2, with Ser-674 to Asp substitution rendering the peptide ineffective.
Conclusions:
- The Ser-674 residue in AC6 is a critical PKA phosphorylation site mediating desensitization to G-alpha-s.
- The AC6 region 660-682 is involved in regulating signal transfer from G-alpha-s.
- Isoform-selective regulatory diversity can arise from modifications within conserved signaling domains, as exemplified by AC6.