Click to download
Residues in extracellular loops 2 and 3 and transmembrane domains 6 and 7 are responsible for the high affinity of salmeterol at the human β2-adrenoceptor. Salmeterol is very selective agonist for the human β2-adrenoceptor (AR) over other β-ARs because of very high binding affinity (Baker 2010). In order to determine key regions in the human β2-AR responsible for this very selective high affinity, mutations were made in the β2-AR whereby each extracellular loop (EL) or each transmembrane region (tm) in turn was mutated such that it contained the amino acid residues of the human β1-AR. Thus β2-EL2 is a β2-AR but with the amino acid sequence of the human β1-AR in the second extracellular loop. 3H-CGP 12177 whole cell binding was then performed in transiently transfected CHO-K1 cells expressing each mutant receptor in turn. Once the main regions were identified (EL or tm domians), each single amino acid in that region was mutated in turn to that of the human β1-AR, and affinity examined (Baker et al., 2008 for molecular biology and binding methods). Four regions were identified as being important for high affinity salmeterol binding – EL2 and EL3, tm6 and tm7. When single amino acid point mutations were examined alone, the most important amino acids were F194V in EL2, R304P and K305D in EL3, H296K in tm6 and Y308F in tm7. The Y308F mutation was found to reduce the affinity of several β2-ligands (see Table, and also fenoterol, clenbuterol and adrenaline) where as other mutations had far greater effect on salmeterol affinity alone. When the mutations were examined in combination, the effects on affinity were found to be additive.
Log KD values from whole cell binding for ligands at the β 1 and β 2 wildtype (WT) receptors, chimeric receptors, and those with single and double point mutations. Values are mean ± s.e.m. of n determinations In conclusion, salmeterol has high selectivity for the human β2-adrenoceptor because of the involvement of specific amino acids in both extracellular loops and within the transmembrane domains. Baker JG (2010) Br. J. Pharmacol. 160: 148-161 Baker et al., (2008) Mol. Pharmacol. 74: 1246-1260
|
