Abstract
We converted Ser-207, located in helix 5 of the beta2-adrenergic receptor, into all other natural amino acids. To quantify receptor activation as a receptor number-independent parameter and directly related to G(s) activation, we expressed the mutants in a G alpha(s)-tethered form. GTP exchange in such constructs is restricted to the fused alpha-subunit and is a linear function of the receptor concentration. Except S207R, all other mutants were expressed to a suitable level for investigation. All mutations reduced the binding affinities of the catechol agonists, epinephrine and isoproterenol, and the extent of reduction was unrelated to the residue ability to form hydrogen bonds. Instead, both enhancements and reductions of affinity were observed for the partial agonist halostachin and the antagonist pindolol. The mutations also enhanced and diminished ligand-induced receptor activation, but the effects were strictly ligand-specific. Polar residues such as Asp and His exalted the activation by full agonists but suppressed that induced by the partial agonists halostachin and dichloroisoproterenol. In contrast, hydrophobic residues such as Ile and Val augmented partial agonist activation. Only Ile and Lys produced a significant increase of constitutive activity. The effects on binding and activity were not correlated, nor did such parameters show any clear correlation with up to 78 descriptors of amino acid physicochemical properties. Our data question the idea that Ser-207 is exposed to the polar crevice in the unbound receptor. They also suggest that the active receptor form induced by a full agonist might be substantially different from that caused by constitutive activation.
Highlights
We converted Ser-207, located in helix 5 of the 2adrenergic receptor, into all other natural amino acids
More recent work [3, 4] revealed that mutagenesis of Ser-203(5.42) can affect ligand binding and activity just like the other two serines and suggests that all three residues may be crucial for catecholamine interaction with the 2AR
Using a double alanine mutant in 5.43 and 5.46 positions (S204A,S207A) we noted that the simultaneous removal of the two residues appears to reduce the rather small constitutive activity of the 2AR, suggesting that the presence of the serine motif in H5 might control the intrinsic equilibrium between active and inactive receptor forms [5]
Summary
Materials—All biochemicals, including nucleotide analogs, were from Sigma. Culture media and fetal calf serum were from Invitrogen. COS7 cells, plated in T25 flasks or 6-wells plates, were washed 48 h after transfection with phosphate-buffered saline, and incubated for 2 min at room temperature with freshly prepared MTSEA at the desired final concentrations. To compare G protein activation across various mutants, membranes were prepared from sets of cells transfected with control pcDNA3 and vectors encoding wild-type and mutant constructs (from 5 up to 18 in each experiment). After protein determinations and the collection of aliquots for the analysis of receptor density (see Bmax above), GTP␥S binding (measured in quadruplicate) was determined for all membranes in the same experiment. The enhancement of nucleotide binding (see “Results” and Fig. 1, C and D) is a linear function of the membrane concentration of fusion protein both in the absence and presence of ligand. The second, (in parenthesis), is the position relative to the most conserved residue of the helix in the macro alignment of class A GPCRs [25]
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