Histamine induces the phosphorylation of a 58 KDa protein in GT1-7 neuronal cells

Exposure to histamine mediates homologous histamine H1 receptor desensitisation by a process involving calcium/calmodulin‐dependent protein kinase II (CaMKII). This suggests a direct involvement of a phosphorylation cascade during desensitisation. Here we investigated histamine‐induced substrate phosphorylation in mouse hypothalamic GT1‐7 neuronal cells. CaMKII activity was detected in GT1‐7 cell extracts incubated with [32P]ATP and autocamtide, a selective CaMKII substrate, which was inhibited by 1‐[N,O‐Bis(5‐isoquinolinesulfonyl)‐N‐methyl‐L‐tyrosyl]‐4‐phenylpiperazine (KN‐62). [32P]orthophosphate‐loaded GT1‐7 cells were exposed to histamine (30 µM, 30 min) with or without KN‐62. Phosphopeptide autoradiograms were generated after 2‐dimensional SDS‐gel (2D‐gel) electrophoresis and the integrated intensity quantified with 2D‐image analysis. Histamine induced the phosphorylation of a 58 kDA phosphoprotein that was prevented by KN‐62. The phosphorylation of this 58 kDa protein suggests a negative feedback step in the signal transduction cascade that results in H1 receptor desensitisation.