Mutation of S and T residues in the third intracellular loop increases intracellular cAMP.

<p><b>A</b>) Schematic identifying potential serine and threonine residues in the third intracellular loop and C-terminus that could be targeted for regulation by phosphorylation. These residues were mutated to alanine to create 3 mutants: ST/A, S1-6A, and ST/A+S1-6A. <b>B</b>) GPR3-HA WT and mutants were transfected into HEK293 cells and harvested 24 hr later for cAMP EIA. (*) indicates a significant increase in cAMP level compared to “WT”. Significance was determined by Repeated Measures ANOVA followed by Dunnett’s Multiple Comparison Test (<i>p</i><0.01). Results are presented as mean ± S.E.M. from 6 separate experiments. <b>C)</b> 24 hr after transfection, cell surface proteins were biotinylated, precipitated, and surface expression of GPR3-HA was detected by Western blotting. 0.5 µg of total lysate was used to detect total GPR3 and β-actin expression. Blot is representative of 3 separate experiments: WT, lane 1; ST/A, lane 2; S1-6A, lane 3; ST/A+S1-6A, lane 4. <b>D–E</b>) Bands corresponding to surface GPR3 and total GPR3 expression were analyzed using densitometry. Densitometric values for total expression of GPR3 WT and mutants were normalized to β-actin (<b>D</b>). The densitometric value for surface GPR3 was divided by the densitometric value for total GPR3 expression and normalized to β-actin to compare the amount of GPR3 at the surface vs. total GPR3 (<b>E</b>). (*) indicates a significant decrease in surface expression of GPR3 mutants compared to “WT”. Significance was determined by One-way ANOVA followed by Newman-Keuls Multiple Comparison Test (<i>p</i><0.05). Results are presented as mean ± S.E.M from 3 separate experiments.