The Effect of Single Nucleotide Polymorphisms on G Protein-Coupled Receptor Kinase Expression and Function

Abstract

The beta-1 adrenergic receptor (&beta1AR) and beta-2 adrenergic receptor (&beta2AR) are two prototypic G protein-coupled receptors (GPCR) targeted in the treatment of a number of diseases, including heart failure, hypertension, and asthma. However, responses to beta adrenergic-targeted therapies vary from individual to individual, making it difficult to predict which patients will benefit from which medication. One potential explanation for this variable response is the presence of genetic variants, such as single nucleotide polymorphisms (SNPs) in the genes encoding beta adrenergic signaling cascade components. Our study focused on G protein-coupled receptor kinases (GRKs), which recognize and phosphorylate agonist-occupied GPCRs, thereby initiating the process of receptor desensitization. We sought to evaluate the role of SNPs in GRKs in two projects. In the first project, we identified SNPs in the promoters of GRK2 and GRK5, two ubiquitously expressed GRKs known to act on both &beta1AR and &beta2AR. We then characterized their effect on promoter activity under basal and ligand-treated conditions. In the second project, we sought to explain the biochemical basis for the reduced response to &beta1AR antagonists seen in patients with certain GRK4 SNPs. We did so by evaluating the interaction of &beta1AR and GRK4 and the effect of SNPs on this interaction. Deep resequencing of African American and Caucasian cohorts revealed multiple SNPs in both GRK2 and GRK5 promoters. These SNPs were organized into haplotypes based on linkage disequilibrium. We used in vitro luciferase reporter assays to determine differences in promoter activity between haplotypes. In the presence of both &beta1AR and &beta2AR, we found variation in promoter activity (as measured by luciferase expression) among haplotypes for both GRK2 and GRK5 under basal conditions. The magnitude of change between basal and ligand-treated conditions was not statistically significant across GRK2 or GRK5 haplotypes in the presence of either &beta1AR or &beta2AR. We did find that only certain GRK5 promoter haplotypes exhibited an increase in activity following antagonist treatment. Further studies, in model systems more relevant to heart failure and asthma/COPD, are necessary to determine the effect of long-term agonist and antagonist exposure on promoter activity among haplotypes. Further studies are also required to determine the effect that variable expression of GRKs has on beta adrenergic receptor desensitization and if this variation is associated with differential response to &beta1AR antagonists and &beta2AR agonists in heart failure and asthma/COPD patients, respectively. In evaluating the interaction of &beta1AR and GRK4, we found evidence of increased phosphorylation of agonist-stimulated &beta1AR in the presence of GRK4. Comparison of &beta1AR desensitization between GRK4 haplotypes did not reveal significant haplotype-specific differences. Future studies will determine functional changes in &beta1AR signaling in the presence of different haplotypes of GRK4. Nevertheless, results of these studies pave the way for further exploration of the biochemical basis for the variable response to &beta1AR antagonists seen among patients with different GRK4 SNPs.