The second extracellular loop of alpha2A-adrenoceptors contributes to the binding of yohimbine analogues

Abstract

Background and purpose: Rodent alpha(2A)-adrenoceptors bind the classical alpha(2)-antagonists yohimbine and rauwolscine with lower affinity than the human alpha(2A)-adrenoceptor. A serine-cysteine difference in the fifth transmembrane helix (TM; position 5.43) partially explains this, but all determinants of the interspecies binding selectivity are not known. Molecular models of alpha(2A)-adrenoceptors suggest that the second extracellular loop (XL2) folds above the binding cavity and may participate in antagonist binding.

Experimental approach: Amino acids facing the binding cavity were identified using molecular models: side chains of residues 5.43 in TM5 and xl2.49 and xl2.51 in XL2 differ between the mouse and human receptors. Reciprocal mutations were made in mouse and human alpha(2A)-adrenoceptors at positions 5.43, xl2.49 and xl2.51, and tested with a set of thirteen chemically diverse ligands in competition binding assays.

Key results: Reciprocal effects on the binding of yohimbine and rauwolscine in human and mouse alpha(2A)-adrenoceptors were observed for mutations at 5.43, xl2.49 and xl2.51. The binding profile of RS-79948-197 was reversed only by the XL2 substitutions.

Conclusions and implications: Positions 5.43, xl2.49 and xl2.51 are major determinants of the species preference for yohimbine and rauwolscine of the human versus mouse alpha(2A)-adrenoceptors. Residues at positions xl2.49 and xl2.51 determine the binding preference of RS-79948-197 for the human alpha(2A)-adrenoceptor. Thus, XL2 is involved in determining the species preferences of alpha(2A)-adrenoceptors of human and mouse for some antagonists.

Figure 1

Molecular structures of (a) yohimbine, (b) rauwolscine, (c) RS-79948-179 and (d) MK-912.

Figure 2

Molecular model of the human α_2A-adrenoceptor, viewed from the extracellular surface. For clarity, only the TM helices are shown (indicated as ribbons). (a) The XL2 domain that forms a β-hairpin (individual strands indicated by green ribbons). (b) The side chains forming the extracellular surface of the binding cavity, xl2.49, xl2.50, xl2.51 and xl2.52 are indicated, and are constrained on top of the cavity by the disulphide bridge connecting xl2.50 (XL2) to 3.25 (TM3).

Figure 3

Schematic comparison of the α_2A-adrenoceptors from (a) human, (b) mouse and rat, and corresponding (c) amino-acid codes according to the Ballesteros and Weinstein (1995) numbering scheme. Amino acids facing the ligand binding cavity in the transmembrane domains and in XL2 are indicated with one-letter codes. Residues differing between the human and mouse receptors are indicated with grey. Only four residues from XL2 (^*) are suggested to face the ligand binding cavity. Amino acids from the expanded binding site close to TM1 suggested by Surgand et al. (2006) are boxed. Cysteines at 3.25 and xl2.50 are connected by a disulphide bridge.

Figure 4

Comparison of the binding affinities of six selected antagonists towards the mouse and human α_2A-adrenoceptors and their mutants. Error bars represent the 95% confidence intervals of 3–5 separate experiments. Statistical significance compared to the wild-type receptor (unpaired t-test): ^*P<0.05, ^**P<0.01, ^***P<0.001.

Figure 5

Docking of yohimbine into the molecular model of the human α_2A-adrenoceptor. The view is from the plane of the membrane through TM6 and TM7 (dark helices); the 7TM bundle is oriented so that TM5 is on the left, TM1 is on the right and XL2 forms the upper surface of the receptor. (a) Overall structure with yohimbine docked within the agonist binding site (in stereo); a close-up view is shown in (b). The left-most ring of (c) rauwolscine, (d) RS-79948-197 and (e) MK-912 was superposed on the ring from yohimbine docked to the human α_2A-adrenoceptor. MK-912, in contrast to yohimbine, rauwolscine and RS-79948-197, clashes with the receptor (especially with W6.48 in TM6) and thus another mode of binding likely takes place. The three side chains mutated in this study are shown: cysteine 5.43 (left of the ligand), glutamate xl2.51 (above the ligand, left) and arginine xl2.49 (above the ligand, right). Carbon atoms are shown in green.

Figure 6

Schematic comparison of the binding cavities of α_2A-adrenoceptors from (a) pig, (b) cow, (c) rabbit, (d) guinea-pig, (e) zebrafish and (f) chicken. The codes used for comparison to the human receptor are as in Figure 2, but amino-acid deletions are shown as triangles. The receptors of species with ‘mouse-like' pharmacology are boxed – with lower affinity for yohimbine.