Addition of a signal peptide sequence to the α _1D- adrenoceptor gene increases the density of receptors, as determined by [ ³H]-prazosin binding in the membranes

Both in mammalian tissues and in transfected cells, only low levels of α _1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the α _1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. In the present study, we created gene constructs for six N-terminally truncated variants of the human α _1D- adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of α _1D-adrenoceptors, observed by [ ³H]-prazosin binding, gradually increased with longer truncations of the N-terminus. This seems to indicate that the long N-terminal tail nonspecifically interferes with receptor translocation to the plasma membrane. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type α _1D-adrenoceptor increased the density of receptor binding sites 10-fold in Neuro2A and COS-7 cells. This indicates that, after the addition of a signal peptide, the long N-terminal tail of the α _1D-adrenoceptor does not interfere with proper translocation of the receptor to the plasma membrane. This, in turn, indicates that the N-terminal tail of the wild-type α _1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. Neither the wild-type α _1D-adrenoceptor (for which the expression level of [ ³H]-prazosin binding sites is low) nor the truncated α _1D-adrenoceptor variant (for which the expression level of [ ³H]-prazosin binding sites is high) showed any constitutive activity in stimulating inositol phosphate accumulation. This indicates that the low expression level of [ ³H]-prazosin binding sites, after transfection with the wild-type α _1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.