Identification of a short sequence highly divergent between β- adrenergic-receptor kinases 1 and 2 that determines the affinity of binding to βγ subunits of heterotrimeric guanine-nucleotide-binding regulatory proteins

A 28-residue peptide (peptide G), derived from the C-terminal (W643- S670) of the β-adrenergic receptor kinase (βARK), was previously identified as the critical domain for binding to the βγ subunits of the heterotrimeric guanine-nucleotide-binding regulatory protein (Gβγ). We observed that the 18-amino-acid core of this domain is poorly conserved between βARK1 and βARK2 and so may provide the basis for differences in Gβγ-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for βARK1 and βARK2, respectively) competitively inhibited Gβγ-activation of the related βARK subtype, confirming the involvement of this region in binding to Gβγ. Peptides P1 and P2 inhibited Gβγ-stimulated activity of both βARKI and βARK2, with P2 being significantly more potent than P1 (IC₅₀ of 179±5 μM for P2 and >500 μM for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC₅₀ = 48 ±5 μM for G2 and 146±8 μM for G1). This relative order of potency G2 > G1 ≃ P2 > P1 was confirmed in a direct Gβγ-binding assay. No binding selectivity for the β1, β2, β3 and β4 Gβ subtypes was observed. The EC₅₀ value for Gβγ-activation of βARK1 was about double of that for βARK2, indicating a higher affinity between Gβγ and βARK2, which is the expected result based on the findings with the peptides. These findings show that the 18-residue peptides P represent the shortest sequence of βARK that can bind to Gβγ and provide a demonstration of a functional difference between the Gβγ binding domains of βARK1 and βARK2.