Constitutive coupling of a naturally occurring human alpha1a-adrenergic receptor genetic variant to EGFR transactivation pathway

Abstract

We previously identified a naturally occurring human SNP, G247R, in the third intracellular loop of the α(1a)-adrenergic receptor (α(1a)-247R) and demonstrated that constitutive expression of α(1a)-247R results in twofold increased cell proliferation compared with WT. In the present study we elucidate molecular mechanisms and signal transduction pathways responsible for increased cell proliferation unique to α(1a)-247R, but not α(1a)-WT, α(1b), or α(1d)AR subtypes. We show that elevated levels of matrix metalloproteinase-7 (MMP7) and a disintegrin and metalloproteinase-12 (ADAM12) in α(1a)-247R-expressing cells are responsible for EGF receptor (EGFR) transactivation, downstream ERK activation, and increased cell proliferation; this pathway is confirmed using MMP, EGFR, and ERK inhibitors. We demonstrate that EGFR transactivation and downstream ERK activation depends on increased shedding of heparin-binding EGF. Finally, we demonstrate that knockdown of MMP7 or β-arrestin1 by shRNAs results in attenuation of proliferation of cells expressing α(1a)-247R. Importantly, accelerated cell proliferation triggered by the α(1a)-247R is serum- and agonist-independent, providing unique evidence for constitutive active coupling to the β-arrestin1/MMP/EGFR transactivation pathway by any G protein-coupled receptor. These findings raise the possibility of a previously unexplored mechanism for sympathetically mediated human hypertension triggered by a naturally occurring human genetic variant.

Fig. 1.

Transmembrane view of a structural model of the human α_1aAR receptor. The α_1aAR contains seven transmembrane domains (TM), TM1 to TM7, four intracellular loops (iL) colored individually, and C-terminal domain (C terminus). The mutation located in the third intracellular loop of the a_1aAR results in substitution of glycine (G) G247 with arginine (R).

Fig. 2.

Constitutive expression of 247R confers unique serum-independent accelerated cell proliferation compared with WT, α_1b or α_1dAR cells. (A) Cells expressing various α₁ARs were synchronized for 24 h in serum-deprived conditions and cultured in 10% FBS or (B) serum-deprived conditions for 24 or 48 h. Data are mean ± SE (SEM) of four independent experiments. *P < 0.05. (C) Flow cytometry analysis of cell-cycle progression of synchronized WT or 247R cells cultured 5 or 24 h. The percentage of cells in proliferative S-phase is indicated.

Fig. 3.

Increased ERK activation in 247R cells is reduced to near normal levels by AG1478 (EGFR), GM6001 (MMP), and PD98059 (MEK) inhibitors. Thymidine incorporation assays of cells cultured in (A) the presence of 10% serum, (B–D) serum-deprived conditions with or without inhibitors, or (D) PE, as indicated. (E) Western blots of cells cultured in the absence (lanes 1 and 4) or presence of AG1478 (lanes 2 and 5) or GM6001 (lanes 3 and 6) with p-ERK or ERK antibodies. (F) Relative intensity of p-ERK protein bands, as determined by densitometric analyses normalized to total ERK. Data are mean ± SEM from three independent experiments. *P < 0.05.

Fig. 4.

Shedding of HB-EGF is increased in 247R cells and is caused by increased levels of Mmp7 and Adam12. (A) HB-EGF transcript level in WT or 247R cells as determined by RT-PCR. (B) Levels of proHB-EGF in WT or 247R cells as determined by ELISA. (C) Growth rate of WT cells cultured in normal or HB-EGF–depleted conditioned medium from WT or 247R cells. Data are mean ± SEM from three independent experiments. (D) Expression of 247R triggers up-regulation of Mmp7 and Adam12 concurrent with reduction in Mmp2. Representative data from one of three independent RT-PCR experiments presented. *P < 0.05.

Fig. 5.

247R Triggered serum and agonist-independent cell proliferation is β-arrestin1/MMP7-dependent and G protein-independent. (A) Mmp7-specific shRNA1 and shRNA4 significantly inhibit Mmp7 expression; GAPDH was used as an internal control. (B) 247R-Induced cell proliferation is inhibited by Mmp7 shRNAs as determined by thymidine incorporation assays. Treatment of cells with 10⁻⁶ M prazosin (C) or 100 ng/mL PTX (D) does not affect cell proliferation. (E) β-Arrestin1 or (F) β-arrestin2–specific shRNAs inhibit the Arrb1 or Arrb2 gene expression, as determined by Western blots; β-actin was used as a loading control. (G) 247R-Induced cell proliferation is inhibited by β-arrestin1 but not β-arrestin2 shRNAs, as determined by thymidine incorporation assays. Data are from one of three independent experiments, each done in triplicates with mean ± SD; *P < 0.05. (H) β-Arrestin1 constitutively interacts with 247R. IP of WT, 247R, or control Rat-1 cells using anti–HA-tag antibody and Western blot with β-arrestin1 antibody. Bands in lanes 5 and 6 represent nonspecific binding of β-arrestin1 to protein A/G Sepharose beads. Representative data from one of three independent experiments are shown. (I) Relative intensity of β-arrestin1 protein bands, as determined by densitometric analyses normalized to the corresponding band intensity of co-IP receptors. Data are mean ± SEM from three independent experiments. **P < 0.01.

Fig. 6.

(A) Initial simplified schematic of 247R-induced serum and agonist independent EGFR transactivation. 247R Triggers β-arrestin1–dependent activation of MMP7 and ADAM12, which shed proHB-EGF to soluble HB-EGF, leading to activation of EGFR. Activated EGFR stimulates the Ras-Raf–MEK-ERK pathway, cell-cycle progression, and cell proliferation. (B) 247R Is colocalized with β-arrestin1. Live WT or 247R cells were labeled with anti-HA antibody (0′), incubated with or without PE (60′), fixed and stained with anti-arrestin1 antibody and visualized with Cy3 (red) or FITC (green) antibodies. Data are representative of three independent experiments. Colocalization is shown in yellow in the merged images (white arrows). (Scale bars, 5 μm.)