Carboxyl terminus-truncated α1D-adrenoceptors inhibit the ERK pathway

Abstract

Human α1D-adrenoceptors are G protein-coupled receptors that mediate adrenaline/noradrenaline actions. There is a growing interest in identifying regulatory domains in these receptors and determining how they function. In this work, we show that the absence of the human α1D-adrenoceptor carboxyl tail results in altered ERK (extracellular signal-regulated kinase) and p38 phosphorylation states. Amino terminus-truncated and both amino and carboxyl termini-truncated α1D-adrenoceptors were transfected into Rat-1, HEK293, and B103 cells, and changes in the phosphorylation state of extracellular signal-regulated kinase was assessed using biochemical and biophysical approaches. The phosphorylation state of other protein kinases (p38, MEK1, and Raf-1) was also studied. Noradrenaline-induced ERK phosphorylation in Rat-1 fibroblasts expressing amino termini-truncated α1D-adrenoceptors. However, in cells expressing receptors with both amino and carboxyl termini truncations, noradrenaline-induced activation was abrogated. Interestingly, ERK phosphorylation that normally occurs through activation of endogenous G protein-coupled receptors, EGF receptors, and protein kinase C, was also decreased, suggesting that downstream steps in the mitogen-activated protein kinase pathway were affected. A similar effect was observed in B103 cells but not in HEK 293 cells. Phosphorylation of Raf-1 and MEK1 was also diminished in Rat-1 fibroblasts expressing amino- and carboxyl-truncated α1D-adrenoceptors. Our data indicate that expression of carboxyl terminus-truncated α1D-adrenoceptors alters ERK and p38 phosphorylation state.

Updates in the function and regulation of α1‐adrenoceptors

“…noradrenaline increased the intracellular calcium in cells expressing Ctail truncated α 1Dadrenoceptors to a similar extent as that seen in cells expressing these receptors with an intact C-tail (Alfonzo-Méndez et al, 2016;Alfonzo-Méndez et al, 2018). In contrast, α 1Dadrenoceptors in which the phosphorylated residues in the third intracellular loop were replaced by non-phosphorylatable amino acids presented a markedly decreased calcium response to noradrenaline .…”

“…To increase membrane expression of this receptor, we used N‐terminally truncated receptors with additional mutations in the third intracellular loop and C‐tail to gain insights on the phosphorylated residues. Among the major findings are the following: noradrenaline increased the intracellular calcium in cells expressing C‐tail truncated α 1D ‐ adrenoceptors to a similar extent as that seen in cells expressing these receptors with an intact C‐tail (Alfonzo‐Méndez et al, ; Alfonzo‐Méndez et al, ). In contrast, α 1D ‐ adrenoceptors in which the phosphorylated residues in the third intracellular loop were replaced by non‐phosphorylatable amino acids presented a markedly decreased calcium response to noradrenaline (Alfonzo‐Méndez et al, ).…”

“…An important additional finding was that although C‐tail‐truncated α 1D ‐ adrenoceptors were able to mobilize calcium in response to noradrenaline, they were mainly detected in intracellular vesicles, and only a minor fraction was detected at the plasma membrane (Alfonzo‐Méndez et al, ; Alfonzo‐Méndez et al, ; Carmona‐Rosas et al, ). The mutation of a motif in the C‐tail of the α 1D ‐ adrenoceptors which interacts with the post synaptic density protein (PSD95)/drosophila disc large tumour suppressor/zonula occludens‐1 protein domain of syntrophins markedly decreased these receptorsat the plasma membrane (Chen, Hague, Hall, & Minneman, ).…”

“…Alfonzo-Méndez et al, 2016;Alfonzo-Méndez et al, 2018;Carmona-Rosas et al, 2018). The mutation of a motif in the C-tail of the α 1Dadrenoceptors which interacts with the post synaptic density protein (PSD95)/drosophila disc large tumour suppressor/zonula…”