Molecular and functional characterization of adipokinetic hormone receptor and its peptide ligands in Bombyx mori

Abstract

Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides, but its signaling pathways remain to be elucidated. In this study, we molecularly characterized the signaling of Bombyx AKH receptor (AKHR) and its peptide ligands in HEK293 cells. In HEK293 cells stably expressing AKHR, AKH1 stimulation not only led to a ligand concentration dependent mobilization of intracellular Ca(2+) and cAMP accumulation, but also elicited transient activation of extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. We observed that AKH receptor was rapidly internalized after AKH1 stimulation. We further demonstrated that AKH2 exhibited high activities in cAMP accumulation and ERK1/2 activation on AKHR comparable to AKH1, whereas AKH3 was much less effective.

Fig. 1

Expression of AKHR in stably transfected HEK293 cells. (A) HEK293 cells stably expressing AKHR–EGFP (GFP) were stained with a membrane plasma probe (DiI) and a nuclei probe (Hoechst 33258). (B) The cell surface expression of the stably transfected HEK293 cells was analyzed by FACS. Stable 293 cells were analyzed for cell surface expression of Flag-AKHR by flow cytometry using the anti-Flag mAb M2. Bars represent the mean fluorescence intensity for cells expressing Flag-AKHR. All data are shown as means ± S.E. from at least three independent experiments.

Fig. 2

AKH-induced cAMP accumulation and intracellular Ca²⁺ mobilization in HEK293 cells stably expressing Flag-AKHR. (A) cAMP accumulation in HEK293 cells transiently co-transfected with CRE-Luc and Flag-AKHR was determined in response to AKH1 treatment (2 µM). (B) Effects of PTX or CTX on AKH-mediated stimulation of cAMP accumulation in Flag-AKHR/CRE/HEK293 cells. HEK293 cells were transiently co-transfected with Flag-AKHR and CRE-Luc, and pre-treated with PTX (100 ng/ml) or CTX (300 ng/ml) overnight prior to incubation with AKH (2 µM) for 4 h. (C) cAMP accumulation in HEK293 cells stably expressing Flag-AKHR/CRE-Luc was assayed in response to different doses of AKH1. Data are expressed as the means ± S.E. (n = 3). (D) Intracellular Ca²⁺ influx in non-transfected 293 cells or 293 cells stably expressing Flag-AKHR was measured in response to different concentrations of AKH1 peptide using the fluorescent Ca²⁺ indicator fura-2. The figures are representative of more than three independent experiments.

Fig. 3

Activation of ERK1/2 by AKH1. (A) AKH1 induce pERK1/2 only in transfected cells, not in controls of the experiment. (B) Time course of AKH-stimulated phosphorylation of ERK1/2 in stable AKHR-expressing HEK293 cells, cells were incubated with 10 µM AKH1 for the indicated times. (C) Concentration-dependent activation of ERK1/2 phosphorylation by AKH1 in HEK293 cells stably expressing Flag-AKHR. Cellular lysates were immunoblotted with phospho-specific (top lane) and non-specific (bottom lane) anti-ERK1/2 antibody, as described in Section 2. The results are representative of at least three independent experiments.

Fig. 4

Time course of AKHR–EGFP internalization induced by AKH1. Cells were incubated with 10 nM AKH at 37 °C for the indicated times, and after washing fixing, were examined by fluorescence microscopy as described in Section 2. The results are representative of three independent experiments.

Fig. 5

Signaling activities and receptor internalization induced by synthesized AKH2 and AKH3 peptides. (A) cAMP accumulation in HEK293 cells stably expressing Flag-AKHR was assayed in response to different doses of AKH2 and AKH3. (B) Activation of ERK1/2 phosphorylation by different doses of AKH2 and AKH3 in HEK293 cells stably expressing AKHR was assayed as described in Section 2. (C) Induction of AKHR–EGFP internalization by AKH2 and AKH3. The stably Flag-AKHR-transfected 293 cells were treated with indicated concentrations of peptide for 45 min at 37 °C. The results are representative of at least three independent experiments.