Role of acidic amino acids in peptide substrates of the .beta.-adrenergic receptor kinase and rhodopsin kinase

Abstract

The beta-adrenergic receptor kinase (beta-ARK) phosphorylates G protein coupled receptors in an agonist-dependent manner. Since the exact sites of receptor phosphorylation by beta-ARK are poorly defined, the identification of substrate amino acids that are critical to phosphorylation by the kinase are also unknown. In this study, a peptide whose sequence is present in a portion of the third intracellular loop region of the human platelet alpha 2-adrenergic receptor is shown to serve as a substrate for beta-ARK. Removal of the negatively charged amino acids surrounding a cluster of serines in this alpha 2-peptide resulted in a complete loss of phosphorylation by the kinase. A family of peptides was synthesized to further study the role of acidic amino acids in peptide substrates of beta-ARK. By kinetic analyses of the phosphorylation reactions, beta-ARK exhibited a marked preference for negatively charged amino acids localized to the NH2-terminal side of a serine or threonine residue. While there were no significant differences between glutamic and aspartic acid residues, serine-containing peptides were 4-fold better substrates than threonine. Comparing a variety of kinases, only rhodopsin kinase and casein kinase II exhibited significant phosphorylation of the acidic peptides. Unlike beta-ARK, RK preferred acid residues localized to the carboxyl-terminal side of the serine. A feature common to beta-ARK and RK was a much greater Km for peptide substrates as compared to that for intact receptor substrates.

Agonist-induced Desensitization of the μ Opioid Receptor Is Determined by Threonine 394 Preceded by Acidic Amino Acids in the COOH-terminal Tail

“…Analysis of phosphorylation of a synthetic peptide based on ␤ 2 AR or ␣ 2 AR sequence showed that negatively charged residues are required for a peptide to serve as a substrate for GRK2 (23). Support for the idea that the Thr 394 serves as the primary recognition site for GRKs, compared with the other potential sites in the carboxyl tail, resulted from the stretch of acidic amino acids in front of it, a mutant receptor was created in which glutamic acid residues at 388, 391, and 393 were replaced by glutamines and Thr 394 was left intact (EQ).…”

“…Interestingly, the feature common to both substrate peptides was the presence of acidic amino acids, mostly glutamic acids, in close proximity to serine and threonine residues (1, 6, 23, 27-31). However, peptides with glutamic acid residues on the carboxyl-terminal side of the serine or threonine were not substrates for GRK2 (23). A peptide with glutamic acid residues on both sides of the serine residue was also a poor substrate.…”

“…A peptide with glutamic acid residues on both sides of the serine residue was also a poor substrate. Furthermore, a peptide that was a good GRK2 substrate became poorly phosphorylated when acidic amino acids at the amino-terminal side of the phosphorylation sites were changed to their neutral counterparts (6,23). This strongly suggests that amino-terminal acidic amino acids relative to the phosphate acceptor group is a requirement for GRK2 recognition.…”

Regulatory mechanisms that modulate signalling by G-protein-coupled receptors

“…Initial studies of substrate determinants for rhodopsin kinase and βARK1\2 used synthetic peptides resembling sequences from the C-terminal tail and the third intracellular loop of various receptors, and identified an Glu\Asp-Xaa-Ser phosphorylation consensus site [64][65][66]. However, intact receptors are better substrates than receptor fragments, and GRKs that are bound to an activated receptor are capable of phosphorylating synthetic peptides which are poor substrates for free kinases [66,67].…”

“…However, intact receptors are better substrates than receptor fragments, and GRKs that are bound to an activated receptor are capable of phosphorylating synthetic peptides which are poor substrates for free kinases [66,67]. Thus GRK activity is greatly enhanced by binding to an activated receptor, and the substrate specificity of GRKs relies on the structure of the activated receptor as well as on the actual amino acid sequence surrounding the phosphorylation site.…”