Adrenergic receptors (ARs) are critical mediators of sympathetic nervous system responses, particularly those involved in cardiovascular homeostasis, such as arteriolar vasoconstriction and cardiac contraction (Graham et al., 1996). Members of the class I or rhodopsin family of G-protein-coupled receptors (GPCRs), ARs consist of nine distinct proteins divided into three subfamilies (α₁-AR, α₂-AR, and β-AR). Like other members of the GPCR superfamily, ARs function as ligand-activated molecular switches coupling catecholamine binding in the extracellular region of the binding pocket, which is formed by the juxtaposition of the seven transmembrane helical segments, to GTP/GDP exchange by the cognate G-protein interacting with their cytoplasmic loops. This topological schema, together with the hydrophilicity of their catecholamine agonists that precludes ready transit across the plasma membrane lipid bilayer, posits that for productive signaling, ARs must be cell surface-expressed. In general, such localization has been found. A possible exception is the α_1D-AR, a subtype initially identified not based on its pharmacological profile but by molecular cloning of its cDNA from a rat hippocampus library (Perez et al., 1991). Indeed, lack of pharmacological data for this subtype initially led to its misidentification as the α_1A-AR (Lomasney et al., 1991), rather than a novel previously uncharacterized subtype (Graham et al., 1996). Although highly conserved in species from fish to mammals, showing 70.5 to 71.3% identity within its transmembrane domains with the corresponding residues of the α_1A- and α_1B-ARs and using similar signaling pathways, the 560-amino acid α_1D-AR differs from its two “siblings” in that it possesses an extra-long (90–95 amino acids) N terminus (Graham et al., 1996). This may contribute to its poor cell-surface expression, although maturational processing by the entire family of GPCRs, which lack a leader sequence, remains poorly understood, with only rhodopsin among the class I family showing relatively robust membrane insertion.

Assembly of rhodopsin begins with the entry of the nascent polypeptide into the endoplasmic reticulum (Khorana, 1992). After high mannose glycosylation, the molecule folds, which involves formation and insertion of the helical segments into the membrane, although this insertion is not coordinated. Thereafter, a structure is formed that includes a disulfide bond between cysteine residues in the juxtamembranous regions of the first and second extracellular loops that are highly conserved in virtually all GPCRs (Khorana, 1992; Graham et al., 1996). This allows alignment of the seven helical segments and establishment of critical interactions between adjacent helical residues, with formation of the mature ligand-binding pocket and concomitantly formation of a specific cytoplasmic domain tertiary structure. Similar paradigms apply for the maturational processing of the β₂-AR (Noda et al., 1994). Unfortunately, biogenesis of the α_1D-AR has been little studied, although the contribution of its long N terminus to its poor cell surface expression is evident from studies showing that expression is markedly enhanced if its N terminus is truncated, or with substitution of its N terminus with that of the α_1B-AR (Fig. 1A) (Pupo et al., 2003; Hague et al., 2004a). Moreover, a reciprocal chimera in which the N terminus of the α_1B-AR is substituted with that of the α_1D-AR shows very poor expression (Hague et al., 2004a). Such poor cell-surface expression is also observed with other GPCRs, such as the calcitonin receptor-like receptor, that require chaperone proteins called receptor activity-modifying proteins (RAMPs) (McLatchie et al., 1998), not only for transport to the cell surface but also for determining their pharmacology and glycosylation state (Foord and Marshall, 1999).

In addition to the low cell-surface expression of the α_1D-AR, which has caused some to question whether the receptor protein even exists in rat tissues (Yang et al., 1997, 1998), it has also been suggested by some to couple less efficiently to its intracellular signaling machinery than the α_1A- and α_1B-ARs (Garcia-Sainz and Villalobos-Molina, 2004), and in the case of the rat α_1D-AR, to exhibit constitutive activity and basal phosphorylation, although such receptor-phosphorylation can be enhanced by agonist-stimulation or phorbol ester treatment (Garcia-Sainz et al., 2001; Garcia-Sainz and Villalobos-Molina, 2004). Given that receptor-phosphorylation may be coupled to desensitization and internalization, the predominant localization of the α_1D-AR in intracellular vesicles may be caused not by tardy biogenesis and sluggish membrane insertion but by constitutive signaling activity, which predisposes it to phosphorylation, desensitization, and internalization (Fig. 1B).

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Fig. 1.

Going to the ball: expression of the α_1D-AR on the cell membrane. The α_1D-AR is poorly expressed on the cell surface. It has been proposed that this is due to the length of the α_1D-AR's N terminus, in that truncation of the N terminus or substitution of it with the N terminus of the α_1B-AR leads to increased cell surface expression (A), a result of constitutive activity of the receptor leading to internalization of the receptor (B), or the need for a chaperone in the form of either the α_1B or β₂-AR to facilitate cell surface expression (C). Dimerization of the α_1D-AR with the α_1B-AR results in a single pharmacological entity that has low affinity for the α_1D-AR selective antagonist BMY-7378; in contrast, dimerization with the β₂-AR or monomeric expression of the α_1D-AR leads to a receptor with high affinity for BMY-7378.

In support of the former scenario, however, Hague et al. (2004) recently demonstrated that not only does the α_1B-AR heterodimerize with the α_1D-AR but also coexpression with the α_1B-AR in a heterologous cell system causes quantitative translocation of the α_1D-AR to the cell surface (Fig. 1C), whereas coexpression with the α_1A-AR does not (Hague et al., 2004b). In this issue of Molecular Pharmacology, the same authors (Hague et al., 2006) now extend these findings and present evidence that, much like the interaction between the calcitonin receptor-like receptor and its RAMP, the α_1B-AR acts as a chaperone to allow not only cell-surface expression of the α_1D-AR but also a novel pharmacological profile (Fig. 1C). However, unlike RAMPs, which are pharmacologically inert, the α_1B-AR is active and, together with the α_1D-AR, forms a novel functional entity that displays enhanced signaling activity. It is noteworthy that the same laboratory describing the effects here of coexpression of the α_1B-AR and α_1D-AR has also recently reported that the β₂-AR can similarly chaperone the α_1D-AR to the cell surface, although in this complex its high affinity for BMY-7378 is unaltered (Fig. 1C) (Uberti et al., 2005).

What evidence do Hague et al. (2006) supply for this elegant transformation from dormant ugly sister to uniquely active Cinderella, so gracefully shod by an α_1B-glass slipper? First, they show that despite confocal microscopy and Western blotting data demonstrating cell-surface localization of the α_1D-AR when coexpressed with the α_1B-AR, a binding site consistent with an α_1D-AP pharmacology cannot be detected. They conclude therefore that heterodimerization with the α_1B-AR, although facilitating the cell surface translocation of the α_1D-AR, nevertheless induces a conformational change in the receptor protein whereby it can no longer bind compounds, such as the α_1D-selective antagonist BMY-7378, with high affinity. Unfortunately, few such subtype-selective compounds are available, and this has been a major factor in limiting our understanding of α₁-AR pharmacology. To obviate this issue, Hague and colleagues cleverly take advantage of a peptide, Rho-conotoxin TIA, recently isolated from the venom of the predatory marine snail Conus tulipa by Sharpe et al. (2001), that acts as an allosteric antagonist of α₁-ARs by binding to sites distinct from the receptors' ligand binding pockets (Sharpe et al., 2003). Using this peptide and an informative mutant previously shown to allow selective recognition of the α_1D-AR (Chen et al., 2004), they demonstrate that despite loss of high-affinity BMY-7378 binding when the α_1B-AR is coexpressed with the α_1D-AR, the latter is nonetheless present on the cell surface, presumably in the form of an α_1B/α_1D heterodimerized complex. Fortunately, the negative influence of the α_1B-AR on high-affinity BMY-7378 binding by the α_1D-AR does not extend to an alteration in its recognition of Rho-conotoxin TIA. They then show that even when the α_1B-AR is coexpressed with an N-terminally truncated α_1D-mutant, which spontaneously traffics to the cell surface but yet can heterodimerize with α_1B-AR (Uberti et al., 2003), a mixed population of cell surface binding sites appears that shows both high and low BMY-7378 affinity, an effect whose magnitude is dependent on the stoichiometry of the two interacting α₁-AR subtypes. Finally, Hague and colleagues (2006) provide evidence that coexpression of the two α₁-AR subtypes results in a unique complex that now signals with greater potency then either subtype alone and that this response is still seen even with coexpression of the α_1D-AR with a signaling incompetent α_1B-AR mutant.

Although one can quibble with some aspects of these studies (for example, for technical reasons, it was not possible to accurately quantitate the stoichiometry of the two α₁-AR subtypes when coexpressed, so a direct effect of one on the transcriptional or translational efficiency of the other cannot be completely excluded), overall, the studies point to a unique, previously unrecognized receptor interaction that has implications for our understanding of the physiology regulated by these two receptor subtypes. For example, knockout studies in which the gene for one or more α₁-AR subtypes has been inactivated have hinted at potential cross-talk between the various subtypes. Thus, in keeping with the present findings, it has been demonstrated that removal of the α_1B-AR leads to the appearance of binding sites with high affinity for BMY-7378 (Daly et al., 2002; Deighan et al., 2005; Hosoda et al., 2005). This suggests that the α_1D-AR is indeed expressed in the vasculature and contributes to maintenance of blood pressure—a contention that is also evident from the reduced blood pressure and impaired vasoconstrictor responses to norepeinephrine of α_1D-knockout mice (Tanoue et al., 2002)— but that its expression is masked by the coincident expression of the α_1B-AR. Of course, this interpretation, and the increasingly robust evidence suggesting that endogenously expressed α₁-ARs heterodimerize, is predicated on more than one α₁-AR subtype being expressed in a single cell, a contention supported by studies of various cell lines (Esbenshade et al., 1993; Bockman et al., 2004), albeit one that has not yet been definitively demonstrated using a single isolated cell.

So what then of the mechanisms by which the α_1B-AR so elegantly envelops the nascent α_1D-AR, enticing her to the cell surface where she dances so vigorously with intracellular signaling partners, and yet smothering her ability to recognize usual antagonist courtiers—well, these questions, although at the heart of the ball, must surely await another evening.