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

Abstract

1 Both in mammalian tissues and in transfected cells, only low levels of a 1D -adrenoceptors are detected in radioligand binding studies. It has been implicated that the comparatively long N-terminal tail of the a 1D -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 a 1D -adrenoceptor. These constructs were used to transfect Neuro2A cells. We show that the density of a 1D -adrenoceptors, observed by [ 3 H]-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 a 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 a 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 a 1D -adrenoceptor, merely by its long length, hinders the first transmembrane helix of the receptor from being a signal anchor.

Heterodimers of α1B- and α1D-Adrenergic Receptors Form a Single Functional Entity

“…It is interesting that we have found that the ␣ 1B -/␣ 1D -AR heterodimer is functionally similar to the GABA B receptor heterodimer. The ␣ 1B -AR serves to promote cell-surface expression of the ␣ 1D -AR (Hague et al, 2004c), possibly by masking an ER retention motif in the ␣ 1D -AR N terminus (Pupo et al, 2003;Hague et al, 2004a;Petrovska et al, 2005). In addition, it seems that within the ⌬12␣ 1B -AR/␣ 1D -AR heterodimer, the ⌬12␣ 1B -AR is primarily responsible for binding ligand, whereas the ␣ 1D -AR couples to G protein activation, but whether this is true for wild-type ␣ 1B -ARs remains to be determined.…”

G Protein-Coupled Receptor Trafficking in Health and Disease: Lessons Learned to Prepare for Therapeutic Mutant Rescue in Vivo

“…Although presently there are no other GPCRs for which this evolved restriction of PME has been documented at the amino acid level, it is possible that this event may be more generally applicable in light of recent reports of other WT receptors that are also inefficiently expressed Uberti et al, 2005;Petrovska et al, 2005;Pietilä et al, 2005;Vandenberghe et al, 2005). It will be necessary to determine whether other proteins follow the same regulatory mechanism.…”

Endogenous N-terminal Domain Cleavage Modulates α1D-Adrenergic Receptor Pharmacodynamics

“…Previous studies revealed truncating the proximal 79 amino acids of the NT liberates ADRA1D from intracellular clusters, facilitates trafficking to the plasma membrane, and enhances functional coupling in response to agonist stimulation (25)(26)(27). Although a useful experimental approach, no data existed suggesting the ADRA1D NT is subjected to post-translational proteolysis in situ or occurred under physiological contexts.…”

“…As a control, the prototype NT ADRA1D mutant, ⌬1-79 ADRA1D, was included in the experimental protocol (Fig. 4B) to facilitate cross-analysis with previous ADRA1D NT studies (25)(26)(27). ␣ 1 -AR agonist efficacies are typically in the rank order of ADRA1A ADRA1B Ͼ ADRA1D in cell culture assays (35).…”

“…SNAP-ADRA1D NT truncation mutants were subcloned with inFusion technology, transfected into HEK293T cells, and imaged with LI-COR Odyssey. ⌬1-58 and ⌬1-79 ADRA1D constructs were included as these mutants were the prototypes first demonstrated to facilitate ADRA1D functional responses in vitro (25)(26)(27). ADRA1D NT deletions with four amino acid increments spanning 79 to 95 were cloned.…”