Characterization of Glucagon-Like Peptide-1 Receptor β-Arrestin 2 Interaction: A High-Affinity Receptor Phenotype

Rasmus Jorgensen, Lene Martini, Thue W. Schwartz, Christian E. Elling
2005 Molecular Endocrinology  
To dissect the interaction between ␤-arrestin (␤arr) and family B G protein-coupled receptors, we constructed fusion proteins between the glucagon-like peptide 1 receptor and ␤arr2. The fusion constructs had an increase in apparent affinity selectively for glucagon, suggesting that ␤arr2 interaction locks the receptor in a high-affinity conformation, which can be explored by some, but not all, ligands. The fusion constructs adopted a signaling phenotype governed by the tethered ␤arr2 with an
more » ... enuated G protein-mediated cAMP signal and a higher maximal internalization compared with wild-type receptors. This distinct phenotype of the fusion proteins can not be mimicked by coexpressing wild-type receptor with ␤arr2. However, when the wild-type receptor was coexpressed with both ␤arr2 and G protein-coupled receptor kinase 5, a phenotype similar to that observed for the fusion constructs was observed. We conclude that the glucagon-like peptide 1 fusion construct mimics the natural interaction of the receptor with ␤arr2 with respect to binding peptide ligands, G protein-mediated signaling and internalization, and that this distinct molecular phenotype is reminiscent of that which has previously been characterized for family A G protein-coupled receptors, suggesting similarities in the effect of ␤arr interaction between family A and B receptors also at the molecular level. (Molecular Endocrinology 19: 812-823, 2005) S EVEN TRANSMEMBRANE (7TM) G protein-coupled receptors interact with heterotrimeric G proteins upon activation. This results in activation of G protein-mediated signal transduction cascades leading to up-or down-regulation of intracellular levels of, for example, cAMP, inositol 1,4,5-triphosphate, or Ca 2ϩ . The termination of the receptor-mediated signaling is regulated by kinase activity phosphorylating the receptor, which leads to recruitment of intracellular scaffolding proteins such as ␤-arrestins (␤arrs) from the cytosol, thereby excluding the receptor from further G protein interaction. Upon interaction with a 7TM receptor, ␤arrs unmask domains that interact with a number of intracellular proteins (for review see Ref. 1) including the clathrin-coated pit components, adaptor protein-2 and clathrin, thereby recruiting the ␤arrinteracting receptor to internalization (2). According to the classical ternary complex model, the receptor has high affinity for agonists when in complex with a G protein. An increasing number of reports on rhodopsin-like family A receptors suggests that when ␤arr interacts with an activated receptor, this likewise results in a stabilization of a distinct highaffinity conformation of the receptor (3-7). Fusion constructs in which the C-terminal tail of the receptor is fused to the N terminus of a G protein or ␤arr have been used to explore the pharmacology of receptors in forced physical proximity to such specific interaction partners (3, 8-10) (for review on receptor-G protein fusions see Ref. 11). Tethering of the receptor to a downstream interaction partner ensures physical proximity between the two potentially interacting proteins, but does not necessarily exclude endogenously expressed proteins from interacting with the receptor (12). In this way the G protein/␤arr may interact with and stabilize transient conformations of the receptors that would not quantitatively recruit G proteins/␤arr unless stabilized by a ligand. Fusion constructs have therefore been used to characterize distinct highaffinity receptor conformations. In the tachykinin NK1 receptor, fusion to ␤arr1 has been shown to create a monocomponent high-affinity receptor phenotype (3). Whereas these are well-established techniques within family A rhodopsin-like receptors, little or no results are available for family B glucagon-like receptors. Most members of the family B 7TM receptor subfamily are promising targets for pharmaceutical intervention. However, these receptors, so far, have proven intractable for classical small molecule drug discovery. Few pharmacological tool compounds therefore exist for
doi:10.1210/me.2004-0312 pmid:15528268 fatcat:djx7ct5oijad7o4n2fozqko7nm