Different phosphorylation patterns regulate α-adrenoceptor signaling and desensitization.

Human α1D-adrenoceptors (α1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/β, phosphorylate α1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α1-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.