Identification of a short sequence highly divergent between beta-adrenergic-receptor kinases 1 and 2 that determines the affinity of binding to betagamma subunits of heterotrimeric guanine-nucleotide-binding regulatory proteins.
A 28-residue peptide (peptide G), derived from the C-terminal (W643-S670) of the beta-adrenergic receptor kinase (betaARK), was previously identified as the critical domain for binding to the betagamma subunits of the heterotrimeric guanine-nucleotide-binding regulatory protein (G betagamma). We observed that the 18-amino-acid core of this domain is poorly conserved between betaARK1 and betaARK2 and so may provide the basis for differences in G betagamma-binding properties. Specific antibodies raised against 18-residue peptides derived from the divergent sequences (peptides P1 and P2 for betaARK1 and betaARK2, respectively) competitively inhibited G betagamma-activation of the related betaARK subtype, confirming the involvement of this region in binding to G betagamma. Peptides P1 and P2 inhibited G betagamma-stimulated activity of both betaARK1 and betaARK2, with P2 being significantly more potent than P1 (IC50 of 179+/-5 microM for P2 and >500 microM for P1). The 28-residue peptides G showed the same relative inhibitory activities (IC50 = 48+/-5 microM for G2 and 146+/-8 microM for G1). This relative order of potency G2 > G1 approximately P2 > P1 was confirmed in a direct G betagamma-binding assay. No binding selectivity for the beta1, beta2, beta3 and beta4 G beta subtypes was observed. The EC50 value for G betagamma-activation of betaARK1 was about double of that for betaARK2, indicating a higher affinity between G betagamma and betaARK2, 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 betaARK that can bind to G betagamma and provide a demonstration of a functional difference between the G betagamma binding domains of betaARK1 and betaARK2.
Chuang
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TT
Istituto di Ricerche Farmacologiche Mario Negri, Consorzio Mario Negri Sud, Santa Maria Imbaro, Italy.
Pompili
E
E
Paolucci
L
L
Sallese
M
M
De Gioia
L
L
Salmona
M
M
De Blasi
A
A
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D001798:Blood Proteins / Q000235:genetics; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D017421:Sequence Analysis; D051552:beta-Adrenergic Receptor Kinases
D001798:Blood Proteins; D010750:Phosphoproteins; C039077:platelet protein P47; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D019204:GTP-Binding Proteins