posted on 2001-12-18, 00:00authored bySonghai Chen, Fang Lin, Ming Xu, Robert M. Graham
We showed previously that Phe303 in transmembrane segment (TM) VI of the α1B-adrenergic
receptor, a highly conserved residue in G-protein-coupled receptors (GPCRs), is critically involved in
receptor-activation and G-protein-coupling [Chen, S. H., Lin, F., Xu, M., Hwa, J., and Graham, R. M.
(2000) EMBO J.19, 4265−4271]. Here, we show that saturation mutagenesis of Phe303 results in a series
of mutants with different levels of constitutive activity for inositol phosphate (IP) signaling. Mutants
F303G and F303N showed neither basal nor agonist-stimulated IP turnover, whereas F303A displayed
increased basal activity but an attenuated maximal response to (−)-epinephrine-stimulation. F303L, on
the other hand, showed all features of a typical constitutively active GPCR with markedly increased basal
activity and increased potency and efficacy of agonist-stimulated IP signaling. All mutants displayed
higher agonist-binding affinities than the wild-type receptor, and by thermal stability studies, those able
to signal showed increased susceptibility to inactivation with an order of sensitivity (F303L > F303A >
WT) directly related to their degree of constitutive activity. Using the substituted cysteine accessibility
method (SCAM) and equilibrium binding studies, we further show that the F303A and F303L mutants
result in TM helical movements that differ in accordance with their degree of constitutive activity. These
findings, therefore, confirm and extend our previous data implicating Phe303 as a key residue coupling
TM helical movements to G-protein-activation.