Effect of GRK and PKA phosphorylation deficient mutations of the β-2 adrenoceptor on antagonist affinity measurements

Jillian G Baker and Stephen J Hill. Institute of Cell Signalling, University of Nottingham, Queen’s Medical Centre, Nottingham, NG7 2UH.

Antagonist affinity measurements at a given receptor are generally considered to be constant for a given receptor-ligand interaction. However previous studies of the Gs-coupled human β2-adrenoceptor have shown that antagonist affinity measurements can vary by up to 10-fold in CRE-gene transcription assays (Baker et al., 2003). One explanation for this is that more efficacious agonists cause both GRK and PKA phosphorylation of the receptor and that this state of the receptor has lower affinity for antagonists (Baker et al., 2003). The aim of this study was to determine whether the agonist-dependent variation in antagonist affinity measurements observed with the wild type human β2-adrenoceptor was also seen with GRK and PKA phosphorylation deficient mutants of the receptor.

Phosphorylation deficient mutants of the human β2-adrenoceptor were generated in pcDNA3.1 by mutating the following residues (Seibold et al., 2000): GRK - (S355A, S356A & S364A), PKA - (S261A, S262A, S345A, S346A) or all seven serine residues (GRK-/PKA-). CHO cells stably expressing a cAMP response element (CRE)-secreted placental alkaline phosphatase (SPAP) reporter gene were secondarily transfected with either the wild-type human β2-adrenoceptor or a phosphorylation deficient mutant and a stable clone selected by dilution cloning. CRE-SPAP production was measured as previously described (Baker et al., 2003).

Isoprenaline stimulated similar concentration-dependent responses at all four receptors (see Table). The log KD values for ICI 118551 and propranolol were also similar for each receptor variant but the Schild slopes were less than unity. Salbutamol produced similar log EC50 values at all four receptors. However the log KD values obtained for ICI 118551 and propranolol with this agonist were circa 10 fold higher than those obtained when isoprenaline was used. Furthermore the Schild slope obtained with salbutamol as agonist were always close to unity.

This study suggests that the phosphorylation status of the GRK and PKA sites of the β2-adrenoceptor is not the reason for the 10-fold different affinity of antagonists measured when isoprenaline and salbutamol are used as agonists.

Table1. log EC50 values for isoprenaline and salbutamol and log KD values for ICI 118551 and propranolol determined in the presence of either isoprenaline or salbutamol (mean ± s.e.m, n)

Baker J.G. et al., (2003). Mol. Pharmacol. 64, 679-688.
Seibold A et al (2000) Mol. Pharmacol.58, 1162-1173.

J.G.B. is a Wellcome Trust Clinician Scientist Fellow