An Acidic Motif within the Third Intracellular Loop of the .alpha.2C2 Adrenergic Receptor Is Required for Agonist-Promoted Phosphorylation and Desensitization

Abstract

The alpha 2C2 adrenergic receptor contains a highly acidic stretch of amino acids (EDEAEEEEEEEEEEEE) within the third intracellular loop. To investigate the role of this region, we utilized site-directed mutagenesis to delete these 16 amino acids as well as to substitute them with glutamine, thereby conserving size but not charge. The wild-type and mutated alpha 2C2 receptors were permanently expressed in CHO cells. Neither substitution nor deletion of this region affected receptor expression, agonist or antagonist binding affinities, guanine nucleotide-sensitive formation of the high-affinity agonist-receptor-G protein complex, or functional coupling of the receptor to Gi. We considered that since alpha 2C2 agonist-promoted desensitization is due to phosphorylation by the beta-adrenergic receptor kinase (or a related kinase), that this region may be important for establishing the acidic mileau required by this kinase. Therefore, the consequences of 30 min of agonist preexposure on subsequent alpha 2C2-mediated inhibition of adenylyl cyclase and on high-affinity agonist binding were determined for the wild-type and these two mutants. The wild-type alpha 2C2 receptor underwent approximately 52% functional desensitization and a approximately 40% loss of high-affinity binding after such exposure. In contrast, deletion and substitution of this acidic stretch of amino acids ablated desensitization as assessed by both approaches. These results correlated with those obtained in whole cell phosphorylation experiments. Cells expressing each receptor were incubated with [32P]orthophosphate and exposed to agonist, and receptors were purified by immunoprecipitation. The deletion and the substitution mutant receptors underwent agonist-promoted phosphorylation at levels of only 44 +/- 5% and 50 +/- 15%, respectively, relative to wild-type alpha 2C2.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of a three-amino acid deletion in the α2B-adrenergic receptor gene on long-term body weight change in Finnish non-diabetic and type 2 diabetic subjects

“…In a study that used site-directed mutagenesis and transfected Chinese hamster ovary cells (CHO), mutant receptors with the glutamic acid repeat element either deleted or replaced with an equal number of charge-neutral glutamine recidues were found to be resistant to short-term, agonist-dependent, phosphorylationmediated desensitization. 21 In another study, mice with a disrupted a 2B -adrenoceptor gene failed to show the typical blood pressure increase after i.v. injection of subtype-nonselective a 2 -adrenoceptor agonists, whereas the centrally mediated hypotensive action of these agents was significantly accentuated.…”

The ζ Isoform of 14-3-3 Proteins Interacts with the Third Intracellular Loop of Different α2-Adrenergic Receptor Subtypes

“…The C-terminal third of the 3i loop of the ␣ 2A AR subtype is implicated in the interaction with ␤-arrestin, a protein that preferentially associates with G protein-coupled receptor kinase-phosphorylated receptors sustaining agonist-elicited homologous desensitization (23). For the ␣ 2 AR subtypes, G protein-coupled receptor kinase phosphorylation sites are in the N-terminal region of the ␣ 2A AR 3i loop (24,25), widely distributed throughout the ␣ 2B AR 3i loop, and presumed to be absent in the ␣ 2C AR 3i loop (13,26).…”

Acidic Amino Acids Flanking Phosphorylation Sites in the M2 Muscarinic Receptor Regulate Receptor Phosphorylation, Internalization, and Interaction with Arrestins

“…Studies with peptide substrates and purified GRKs have suggested that acidic amino acids act to direct GRKmediated phosphorylation of specific Ser/Thr in GPCRs (16). Studies with the ␣ 2 C2 adrenergic receptor demonstrated that acidic amino acids are important in the agonist-dependent phosphorylation of this receptor (40). Removal of the large cluster of acidic residues in this receptor decreased the extent of phosphorylation of this receptor approximately 50% (40).…”

“…Studies with the ␣ 2 C2 adrenergic receptor demonstrated that acidic amino acids are important in the agonist-dependent phosphorylation of this receptor (40). Removal of the large cluster of acidic residues in this receptor decreased the extent of phosphorylation of this receptor approximately 50% (40). However, little is known about the role of acidic amino acids in directing phosphorylation of specific sites in other GPCRs in a cellular context.…”