Differential regulation of histamine H1 receptor-mediated ERK phosphorylation by Gq proteins and arrestins

by Selleckchem | Jul 02 2023

Gq protein-coupled histamine H1 receptors play crucial roles in allergic and inflammatory reactions, in which the phosphorylation of extracellular signal-regulated kinase (ERK) appears to mediate the production of inflammatory cytokines. ERK phosphorylation is regulated by G protein- and arrestin-mediated signal transduction pathways. Here, we aimed to explore how H1 receptor-mediated processes of ERK phosphorylation might be differentially regulated by Gq proteins and arrestins. For this purpose, we evaluated the regulatory mechanism(s) of H1 receptor-mediated ERK phosphorylation in Chinese hamster ovary cells expressing Gq protein- and arrestin-biased mutants of human H1 receptors, S487TR and S487A, in which the Ser487 residue in the C-terminal was truncated and mutated to alanine, respectively. Immunoblotting analysis indicated that histamine-induced ERK phosphorylation was prompt and transient in cells expressing Gq protein-biased S487TR, whereas it was slow and sustained in cells expressing arrestin-biased S487A. Inhibitors of Gq proteins (YM-254890) and protein kinase C (PKC) (GF109203X), and an intracellular Ca2+ chelator (BAPTA-AM) suppressed histamine-induced ERK phosphorylation in cells expressing S487TR, but not those expressing S487A. Conversely, inhibitors of G protein-coupled receptor kinases (GRK2/3) (cmpd101), β-arrestin2 (β-arrestin2 siRNA), clathrin (hypertonic sucrose), Raf (LY3009120), and MEK (U0126) suppressed histamine-induced ERK phosphorylation in cells expressing S487A, but not those expressing S487TR. These results suggest that H1 receptor-mediated ERK phosphorylation might be differentially regulated by the Gq protein/Ca2+/PKC and GRK/arrestin/clathrin/Raf/MEK pathways to potentially determine the early and late phases of histamine-induced allergic and inflammatory responses, respectively.

Comments:

The study you described aimed to investigate the regulatory mechanisms of histamine H1 receptor-mediated phosphorylation of extracellular signal-regulated kinase (ERK) in Chinese hamster ovary cells expressing Gq protein- and arrestin-biased mutants of human H1 receptors.

The researchers used two mutants of the human H1 receptor: S487TR and S487A. In the S487TR mutant, the Ser487 residue in the C-terminal was truncated, while in the S487A mutant, it was mutated to alanine. These mutants allowed the researchers to selectively study the effects of Gq proteins and arrestins on H1 receptor-mediated ERK phosphorylation.

Immunoblotting analysis showed that histamine-induced ERK phosphorylation had distinct characteristics in cells expressing the different mutants. In cells expressing the Gq protein-biased S487TR mutant, ERK phosphorylation was prompt and transient. On the other hand, in cells expressing the arrestin-biased S487A mutant, ERK phosphorylation was slow and sustained.

To further understand the regulatory mechanisms involved, the researchers tested various inhibitors targeting different signaling pathways. In cells expressing S487TR, inhibitors of Gq proteins (YM-254890), protein kinase C (PKC) (GF109203X), and an intracellular calcium chelator (BAPTA-AM) suppressed histamine-induced ERK phosphorylation. In contrast, these inhibitors had no effect in cells expressing S487A. This suggests that Gq protein/Ca2+/PKC signaling is involved in the prompt and transient ERK phosphorylation observed in the Gq protein-biased mutant.

Conversely, in cells expressing S487A, inhibitors of G protein-coupled receptor kinases (GRK2/3) (cmpd101), β-arrestin2 (β-arrestin2 siRNA), clathrin (hypertonic sucrose), Raf (LY3009120), and MEK (U0126) suppressed histamine-induced ERK phosphorylation. These inhibitors had no effect in cells expressing S487TR. This indicates that the GRK/arrestin/clathrin/Raf/MEK pathway is involved in the slow and sustained ERK phosphorylation observed in the arrestin-biased mutant.

In summary, the study suggests that H1 receptor-mediated ERK phosphorylation is differentially regulated by Gq proteins and arrestins. The Gq protein/Ca2+/PKC pathway appears to be responsible for the prompt and transient phase of ERK phosphorylation, while the GRK/arrestin/clathrin/Raf/MEK pathway is involved in the slow and sustained phase. These findings suggest that the differential regulation of ERK phosphorylation by these pathways may contribute to the early and late phases of histamine-induced allergic and inflammatory responses, respectively.