Margaret G. Eason

Identification of a Gs Coupling Domain in the Amino Terminus of the Third Intracellular Loop of the α2A-Adrenergic Receptor EVIDENCE FOR DISTINCT STRUCTURAL DETERMINANTS THAT CONFER Gs VERSUS Gi COUPLING

α2-Adrenergic receptors (α2AR) functionally couple not only to Gi but also to Gs. We investigated the amino-terminal portion of the third intracellular loop of the human α2AAR (α2C10) for potential Gs coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the α2AAR with the analogous sequence from the 5-HT1A receptor (a Gi-coupled receptor) and the β2AR (a Gs-coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation. In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated α2C10-mediated stimulation of adenylyl cyclase activity, while α2C10-mediated inhibition was only moderately affected. This suggested that this region is important for Gs coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218-235 with 5-HT1A receptor sequence entirely ablated agonist-promoted Gs coupling, as compared with a 338 ± 29% stimulation of adenylyl cyclase activity observed with the wild-type α2C10. In contrast, Gi coupling for this mutant remained fully intact (57 ± 2% versus 52 ± 1% inhibition for wild-type α2C10). Similar substitution with β2AR sequence had no effect on Gi coupling but did reduce Gs coupling. Two additional mutated α2C10 containing smaller substitutions of the amino-terminal region with 5-HT1A receptor sequence at residues 218-228 or 229-235 were then studied. While Gi coupling remained intact with both mutants, Gs coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese hamster fibroblasts (which exclusively display α2AR-Gi coupling) and COS-7 cells (which exclusively display α2AR-Gs coupling). Thus, a critical determinant for Gs coupling is contained within 11 amino acids(218-228) of the amino-terminal region of the third intracellular loop localized directly adjacent to the fifth transmembrane domain. Taken together, these studies demonstrate the presence of a discrete structural determinant for agonist-promoted α2AR-Gs coupling, which is distinct and separable from the structural requirements for α2AR-Gi coupling.

Role of βARK in Long-Term Agonist-Promoted Desensitisation of the β2-Adrenergic Receptor

Phosphorylation of the β2-adrenergic receptor (β2AR) is the initial event that underlies rapid agonist-promoted desensitisation. However, the role of phosphorylation in mediating long-term β2AR desensitisation is not known. To investigate this possibility, we performed intact cell phosphorylation studies with COS-7 cells transiently expressing an epitope tagged wild-type β2AR and found that receptor phosphorylation in cells treated with 1 μM isoproterenol for 24 h was ∼4-fold over the basal state. This finding suggested that persistent phosphorylation of the receptor might contribute to functional long-term desensitisation which we further explored with mutated β2AR lacking the determinants of phosphorylation by the βAR kinase (βARK), PKA or both. In CHW cells expressing the WT β2AR, pretreatment with 1 μM isoproterenol for 24 h reduced the isoproterenol-stimulated cAMP response by 82 ± 5%. Substitution of the PKA sites with alanines had no effect on the extent of desensitisation (77 ± 6%, P = NS compared to WT). In contrast, desensitisation was only 49 ± 4% (P < 0.001 compared to WT) when the βARK sites were similarly substituted. Removal of both the βARK and PKA sites impaired desensitisation to the same extent as the βARK mutant. The extent of receptor loss (downregulation) was the same among all of the cell lines used and therefore could not account for the observed differences in desensitisation. Cellular βARK activity, assessed by a rhodopsin phosphorylation assay, was equivalent in all cell lines and was unaffected by agonist treatment. PKA activity, however, was dynamically regulated, increasing 4-fold over basal levels after 15 min of isoproterenol and returning to near basal levels after 24 h. The lower level of PKA activity after long-term agonist exposure may therefore have contributed to the apparent lack of effect of removing PKA sites. Nonetheless, long-term desensitisation was clearly attenuated with β2AR lacking βARK phosphorylation sites. These findings show that in addition to its role in regulating short-term desensitisation, βARK-mediated phosphorylation is an important mechanism underlying long-term desensitisation of the β2AR as well.

Phosphorylation of Ser360 in the third intracellular loop of the α2A-adrenoceptor during protein kinase C-mediated desensitization

The alpha2A-adrenoceptor undergoes desensitization in response to protein kinase C activation. Using mutagenesis and recombinant expression we sought to determine the specific sites within the receptor which are phosphorylated by protein kinase C and are responsible for this desensitization. Ser/Thr in the third intracellular loop were substituted with Ala to create mutant receptors T272A, S258A, S324A and S360A. These mutations had no effect on ligand binding or functional coupling to inositol phosphate accumulation and intracellular Ca2+ release. Three of the four mutant receptors displayed wild-type desensitization (approximately 60% loss of function) in response to 0.1 microM phorbol-12-myristate-13-acetate (PMA) exposure for 2 min. However, the S360A mutant had only approximately 24% desensitization. In whole cell phosphorylation studies, S360A failed to undergo detectable PMA promoted phosphorylation. We conclude that protein kinase C-mediated desensitization of alpha2A-adrenoceptor function is primarily due to phosphorylation of Ser at amino acid 360. This thus represents one mechanism by which these receptors undergo regulation by heterologous means, such as pathologic processes which activate protein kinase C or crosstalk with other receptors.