Purification and characterization of β-adrenergic receptor mRNA-binding proteins

Abstract

β-Adrenergic receptors (β-ARs), like other G-protein-coupled receptors, can undergo post-transciptional regulation at the level of mRNA stability. In particular, the human β1- and β2-ARs and the hamster β2- AR mRNA undergo β-agonist-mediated destabilization. By UV cross-linking, we have previously described an ~M(r) 36,000 mRNA-binding protein, βARB, that binds to A/C+U-rich nucleotide regions within 3'-untranslated regions. Further, we have demonstrated previously that βARB is immunologically distinct from AUF1/heterogeneous nuclear ribonucleoprotein (hnRNP) D, another mRNA-binding protein associated with destabilization of A+U-rich mRNAs (Pende, A., Tremmel, K. D., DeMaria, C. T., Blaxall, B.C., Minobe, W., Sherman, J. A., Bisognano, J., Bristow, M. R., Brewer, G., and Port, J. D. (1996) J. Biol. Chem. 271, 8493-8501). In this report, we describe the peptide composition of βARB. Mass spectrometric analysis of an ~M(r) 36,000 band isolated from ribosomal salt wash proteins revealed the presence of two mRNA-binding proteins, hnRNP A1, and the elav-like protein, HuR, both of which are known to bind to A+U-rich nucleotide regions. By immunoprecipitation, HuR appears to be the biologically dominant RNA binding component of βARB. Although hnRNP A1 and HuR can both be immunoprecipitated from ribosomal salt wash proteins, the composition of βARB (HuR alone versus HuR and hnRNP A1) appears to be dependent on the mRNA probe used. The exact role of HuR and hnRNP A1 in the regulation of β-AR mRNA stability remains to be determined.