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

Publisher Website
Google Scholar
Abstract
A 28-residue peptide (peptide G), derived from the C-terminal (W643-S670) of the beta-adrenergic receptor kinase (beta ARK), was previously identified as the critical domain for binding to the beta gamma subunits fo the heterotrimeric guanine-nucleotide-binding regulatory protein (G beta gamma). We observed that the 18-amino-acid core of this domain is poorly conserved between beta ARK1 and beta ARK2 and so may provide the basis for differences in G beta gamma-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for beta ARK1 and beta ARK2, respectively) competitively inhibited G beta gamma-activation of the related beta ARK subtype, confirming the involvement of this region in binding to G beta gamma. Peptides P1 and P2 inhibited G beta gamma-stimulated activity of both beta ARK1 and beta ARK2, with P2 being significantly more potent than P1 (IC50 of 179 +/- 5 mu M for P2 and > 500 mu M for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC50 = 48 +/- 5 mu M for G2 and 146 +/- 8 mu M for G1). This relative order of potency G2 > G1 approximate to P2 > P1 was confirmed in a direct G beta gamma-binding assay. No binding selectivity for the beta 1, beta 2, beta 3 and beta 4 G beta subtypes was observed. The EC50 value for G beta gamma-activation of beta ARK1 was about double of that for beta ARK2, indicating a higher affinity between G beta gamma and beta ARK2, 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 beta ARK that can bind to G beta gamma and provide a demonstration of a functional difference between the G beta gamma binding domains of beta ARK1 and beta ARK2