Bombyx Adipokinetic Hormone Receptor Activates Extracellular Signal-Regulated Kinase 1 and 2 via G Protein-Dependent PKA and PKC but β-Arrestin-Independent Pathways

Abstract

Neuropeptides of the adipokinetic hormone (AKH) family are among the best studied hormone peptides. They play important roles in insect hemolymph sugar homeostasis, larval lipolysis, and storage-fat mobilization. Mechanistic investigations have shown that, upon AKH stimulation, adipokinetic hormone receptor (AKHR) couples to a Gs protein and enhances adenylate cyclase activity, leading to intracellular cAMP accumulation. However, the underlying molecular mechanism by which this signaling pathway connects to extracellular signal-regulated kinase 1/2 (ERK1/2) remains to be elucidated. Using HEK293 cells stably or transiently expressing AKHR, we demonstrated that activation of AKHR elicited transient phosphorylation of ERK1/2. Our investigation indicated that AKHR-mediated activation of ERK1/2 was significantly inhibited by H-89 (protein kinase A inhibitor), Go6983, and GF109203X (protein kinase C inhibitors) but not by U73122 (PLC inhibitor) or FIPI (PLD inhibitor). Moreover, AKHR-induced ERK1/2 phosphorylation was blocked by the calcium chelators EGTA and BAPTA-AM. Furthermore, ERK1/2 activation in both transiently and stably AKHR-expressing HEK293 cells was found to be sensitive to pretreatment of pertussis toxin, whereas AKHR-mediated ERK1/2 activation was insensitive to siRNA-induced knockdown of β-arrestins and to pretreatment of inhibitors of EGFR, Src, and PI3K. On the basis of our data, we propose that activated AKHR signals to ERK1/2 primarily via PKA- and calcium-involved PKC-dependent pathways. Our current study provides the first in-depth study defining the mechanisms of AKH-mediated ERK activation through the Bombyx AKHR.

Identification and Functional Characterization of Two Orphan G-protein-coupled Receptors for Adipokinetic Hormones from Silkworm Bombyx mori

“…The influx of extracellular Ca 2ϩ via G q -mediated phospholipase C is also essential for the adipokinetic effect (36,37). Our previous studies have demonstrated that agonist-activated AKHRs signal the ERK1/2 pathway via PKA-and calcium-involved PKC-dependent pathways (18). We have further demonstrated that AKHRs are rapidly internalized in a dose-and time-dependent manner via the clathrin-coated pit pathway upon agonist stimulation, and internalized receptors were slowly recovered to the cell surface after the removal of AKH peptides (26).…”

“…In addition, AKH activates phospholipase C to induce the release of Ca 2ϩ from intracellular Ca 2ϩ stores (16,17). Our previous studies have demonstrated that AKH stimulation not only led to a ligand concentration-dependent mobilization of intracellular Ca 2ϩ and cAMP accumulation but also elicited PKA and PKC pathway-mediated transient activation of the extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathways in HEK293 cells stably expressing AKHR (18). We have further demonstrated that AKH2 exhibits comparable activities in intracellular cAMP accumulation to AKH1, but Bombyx AKH3 was much less effective in activating Bombyx AKHR, strongly implying that there is more likely other intrinsic AKHRs that exist as a high affinity receptor for AKH3 in Bombyx (19).…”

Valproic Acid Influences MTNR1A Intracellular Trafficking and Signaling in a β-Arrestin 2-Dependent Manner

“…The mouse neural crest-derived cell line, Neuro-2a, and Human Embryonic Kidney 293 cells, HEK-293, were used as described previously [23]. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA).…”

“…MTNR1A (in pFLAG-CMV-3) and β-arrestin 2 (in pEGFP-N1) was a kind gift of Zhou Naiming (Zhejiang University, China) [23]. MTNR1A was modified by PCR and subcloned into EGFP, ECFP, or mCherry.…”

More than two decades of research on insect neuropeptide GPCRs: an overview

“…The Drosophila AKHR was the first to be cloned and was deorphanized by making use of the electrophysiological assay (Park et al, 2002), and its characterization was confirmed by Staubli et al (2002) using a bioluminescence assay. Later, AKHRs were also identified and characterized in other insect species: Periplaneta americana (Hansen et al, 2006; Wicher et al, 2006a), A. gambiae (Belmont et al, 2006), B. mori (Staubli et al, 2002; Zhu et al, 2009; Huang et al, 2010), and T. castaneum (Li, unpublished data). Two putative AKHR variants have been predicted in A. aegypti (Kaufmann et al, 2009) and one AKHR is identified in the Apis genome (Hauser et al, 2006).…”