Different phosphorylation patterns regulate α1D-adrenoceptor signaling and desensitization

Abstract

Human α-adrenoceptors (α-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 α-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 α-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 α-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 α-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.

Updates in the function and regulation of α1‐adrenoceptors

“…Schematic representation of the substrates for phosphorylation currently identified in the three human α 1 ‐adrenoceptors (AR) subtypes (large red, yellow, and blue circles). Phosphorylated residues in the α 1A ‐ and α 1D ‐ adrenoceptors were identified by MS (Alcantara‐Hernandez et al, ; Alfonzo‐Méndez et al, ), whereas for the α 1B ‐adrenoceptor, the residues were identified by site‐directed mutagenesis and functional analysis of the hamster orthologue (Diviani et al, ; in parenthesis are the sites defined for the hamster receptor). Below the diagram are the sequence alignments of the third intracellular loop and the carboxyl terminus (C‐tail) of the three human α 1 ‐adrenoceptor subtypes.…”

“…The phosphorylation sites of α 1A ‐ and α 1D ‐adrenoceptors were only recently identifed (Alcantara‐Hernandez et al, ;Alfonzo‐Méndez et al, ;Carmona‐Rosas et al, ). These adrenoceptors are phosphorylated in both the third intracellular loop and C‐tail.…”

“…For α 1A ‐ adrenoceptors, there were differences in the phosphorylation patterns induced by noradrenaline and oxymetazoline (Alcantara‐Hernandez et al, ). Not surprisingly, in silico analysis indicated the possible roles for GRK and PKC isoforms, but other kinases may also be involved (Alcantara‐Hernandez et al, ; Alfonzo‐Méndez et al, ). It is interesting that the phosphorylation sites detected for the α 1 ‐adrenoceptor subtypes are not conserved among them and in only a few cases are the sites are conserved between two subtypes (α 1A S250/α 1D S323; α 1A S258/α 1D S334; α 1A S407/α 1D T507; see Figure ).…”

“…The data indicated that the third loop is important for signalling through G‐protein coupling, as proposed for many GPCRs, and raises the possibility that phosphorylation of these residues might be important for receptor G‐protein activation. The possibility that the C‐tail could also participate in calcium signalling was not discarded, because in cells expressing C‐tail‐truncated α 1D ‐ adrenoceptors with the third intracellular loop mutated, the calcium response to noradrenaline was decreased even further (Alfonzo‐Méndez et al, ; Carmona‐Rosas et al, ). ERK 1/2 phosphorylation in cells expressing these mutants was also studied.…”

“…ERK 1/2 phosphorylation in cells expressing these mutants was also studied. In cells expressing the control receptor, noradrenaline induced a rapid ERK 1/2 phosphorylation that decreased at 30 and 60 min, whereas in cells with the different mutants, the response was also rapid but either decreased much more slowly or remained elevated during the entire 60 min (Alfonzo‐Méndez et al, ; Carmona‐Rosas et al, ). This indicates that some of the phosphorylated residues, in both the third intracellular loop and C‐tail, might participate in turning off the signal in the MAPK pathway (Alfonzo‐Méndez et al, ; Carmona‐Rosas et al, ).…”