Addition of a signal peptide sequence to the alpha1D-adrenoceptor gene increases the density of receptors, as determined by [3H]-prazosin binding in the membranes

Abstract

1. Both in mammalian tissues and in transfected cells, only low levels of alpha1D-adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the alpha1D-adrenoceptor is responsible for the inefficient surface expression of the receptor. 2. In the present study, we created gene constructs for six N-terminally truncated variants of the human alpha1D-adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of alpha1D-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. 3. The addition of a 16 amino acids long signal peptide to the N-terminus of the wild-type alpha1D-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 alpha1D-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 alpha1D-adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor. 4. Neither the wild-type alpha1D-adrenoceptor (for which the expression level of [3H]-prazosin binding sites is low) nor the truncated alpha1D-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 alpha1D-adrenoceptor, is not caused by constitutive activity of the receptor and subsequent receptor downregulation.

Figure 1

Saturation curves for [³H]-prazosin on membranes after transient or stable transfections of Neuro2A cells with different α_1D-AR constructs. Nonspecific binding was defined by 2 μM of metitepine.

Figure 2

Dose–response curves for the α₁-agonist phenylephrine on [³H]-inositol phosphate accumulation in Neuro2A cells stably transfected with the wt α_1A-, α_1B-, and α_1D-adrenoceptors, or with the truncated α_1D-AR variants Δ1–17α_1D and Δ1–58α_1D.

Figure 3

Schematic illustration of α_1D-adrenoceptor surface expression in transfected cells. The wt α_1D-AR is poorly translocated to the plasma membrane (a). Truncated α_1D-AR variant (b) and the α_1D-AR supplemented with a cleavable signal peptide (c) are well expressed in the membranes. Coexpression of α_1D-AR with the α_1B-subtype increases surface expression of α_1D-adrenoceptors (d). The well-expressed receptors (b–d) show a six to 10-fold increase in the density of binding sites in the plasma membrane compared to the wt α_1D-AR. Incubation of α_1D-AR-expressing cells with the α₁-antagonist prazosin induces an increased density of receptors in the plasma membrane (e). The figure is based on results reported in the present study (c), and in Pupo et al. (2003) (a and b), Hague et al. (2004b) (d), and McCune et al. (2000) (e).