An investigation into the regulatory mechanisms associated with the recombinant human 5-hydroxytryptamine1a (5-HT1a) receptor

Abstract

Classically, acute agonist challenge results in a concomitant loss of G protein-coupled receptor signalling function and high affinity membrane ligand binding sites.

Expression of the human 5-HT_1A receptor in CHO-K1 cells allowed the study of a homogeneous receptor population expressed at high density. Exposure of CHO-K1 cells expressing the cloned 5-HT_1A receptor to 1μM- 8-hydroxyl-2-(di-n-propylamino)-tetralin (8-hydroxy-DPAT) resulted in receptor internalisation (20% of receptors after 20 minutes) to an undefined intracellular compartment. Receptor internalisation was found to be sensitive to, hypertonic sucrose (0.7-0.8M) and Concanavalin A (0.5-1mg/ml), both of which are inhibitors of clathrin-mediated endocytosis. Removal of 8-hydroxy-DPAT from the incubation medium allowed 5-HT_1A receptors to recycle back to the plasma membrane, this process occurred in the presence of the protein synthesis inhibitor cycloheximide and in a time dependent fashion (40-50 minutes). Mutagenesis of the putative PKC phosphorylation motifs, KRT₁₄₉APR and effect upon receptor binding characteristics however, complete abolition of receptor redistribution in response to acute agonist challenge was observed. In a similar manner conservative and radical mutation of the internalisation motif NPVIY (Y₄₀₀F and Y₄₀₀A respectively) had no effect on receptor binding characteristics but they did abolish receptor internalisation in response to short term agonist incubation.

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