Different Phosphorylation Sites Regulate α1D‐Adrenergic Receptor Function

Human α1D‐adrenergic receptors (α1D‐AR) are seven transmembrane‐spanning proteins that mediate the actions of noradrenaline in homeostasis maintenance as well as the physiology of hypertension and benign prostatic hyperplasia. Although several studies have demonstrated that α1D‐AR is a phosphoprotein, the phosphorylation sites and the specific roles that they play in the function and regulation of this receptor, are currently unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified for the first time distinct phosphorylation patterns on α1D‐AR during noradrenaline (homologous) and non‐related agents‐mediated (heterologous) desensitization. We were able to identify that G protein‐coupled receptor kinase‐2 (GRK2) and protein kinase C α/β (PKC α/β) phosphorylate α1D‐AR during these processes. Phosphorylation of the receptor's intracellular domains, such the intracellular loop 3 or the carboxyl tail, was enough to altered calcium desensitization signals, mitogen activated protein kinases (MAPK) pathway and regulate receptor internalization, suggesting that modular regulatory mechanisms heavily rely on the phosphorylation bar code and the receptor's intracellular domains. Subsequently, we generated six specific non‐phosphorylatable α1D‐AR mutants to delve into the functions of the phosphorylation sites located only at the receptor's carboxyl tail. We found clear differences in the phosphorylation pattern of each mutant during noradrenaline and non‐related agents‐stimulation. Moreover, we analyzed the activation of the MAPK and AKT pathways as well as intracellular calcium mobilization. Modification of any of these phosphorylation sites altered the response when compared to the wild type, but no clear differences were observed among the different α1D‐AR mutants. Interestingly, seven specific phosphorylation sites clustered into two groups in the carboxyl tail, regulate the α1D‐AR subcellular location. This was confirmed when these two clusters were mutated with negative charged residues and the α1D‐AR was located again at the plasma membrane. These data suggest that different phosphorylation sites located in the carboxyl tail regulate the α1D‐AR functions and regulation at different levels. This knowledge highly contribute to unveil new strategies to improve current pharmacological treatments and to avoid drug side effects during sustained administration. Support or Funding Information Partially supported by Grants from CONACYT (253156 and 882) and DGAPA (IN200915). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .