The Adaptor Complex 2 Directly Interacts with the α1b-Adrenergic Receptor and Plays a Role in Receptor Endocytosis

Abstract

Using the yeast two-hybrid system, we identified the 2 subunit of the clathrin adaptor complex 2 as a protein interacting with the C-tail of the ␣ 1b -adrenergic receptor (AR). Direct association between the ␣ 1b -AR and 2 was demonstrated using a solid phase overlay assay. The ␣ 1b -AR/ 2 interaction occurred inside the cells, as shown by the finding that the transfected ␣ 1b -AR and the endogenous 2 could be coimmunoprecipitated from HEK-293 cell extracts. Mutational analysis of the ␣ 1b -AR revealed that the binding site for 2 does not involve canonical YXX⌽ or dileucine motifs but a stretch of eight arginines on the receptor C-tail. The binding domain of 2 for the receptor C-tail involves both its N terminus and the subdomain B of its C-terminal portion. The ␣ 1b -AR specifically interacted with 2 , but not with the 1 , 3 , or 4 subunits belonging to other AP complexes. The deletion of the 2 binding site in the C-tail markedly decreased agonist-induced receptor internalization as demonstrated by confocal microscopy as well as by the results of a surface receptor biotinylation assay. The direct association of the adaptor complex 2 with a G protein-coupled receptor has not been reported so far and might represent a common mechanism underlying clathrin-mediated receptor endocytosis.Desensitization to the effects of hormones and neurotransmitters is a fundamental regulatory mechanism of G proteincoupled receptor (GPCR) 1 function defined by a specific loss of responsiveness for those receptors that have been repeatedly stimulated by the agonist. Receptor desensitization results from the combination of multiple biochemical events occurring at different time frames: receptor-G protein uncoupling in response to receptor phosphorylation (seconds to minutes), internalization or endocytosis of cell surface receptors to intracellular compartments (minutes), and down-regulation of the total pool of receptors due to their decreased synthesis and/or increased degradation (hours) (1). A prominent role in homologous desensitization of GPCRs is played by G protein-coupled receptor kinases. Once the receptor is occupied by the agonist, it is recognized by the G protein-coupled receptor kinases and becomes phosphorylated (2). The subsequent uncoupling of the receptor from the G protein is then mediated by arrestin proteins, which preferentially bind to the agonist-occupied phosphorylated receptor (3). However, during the last decade, ␤-arrestins have emerged as key regulatory molecules controlling various steps of receptor desensitization. Beyond their role in physical uncoupling of GPCRs from the G proteins (3), it has been demonstrated that ␤-arrestins target GPCRs to the endocytic machinery (4). In fact, it is believed that for those GPCRs that internalize in a clathrin-dependent manner, like the ␤ 2 -adrenergic receptor (AR), targeting of the receptor-␤-arrestin complexes to clathrin-coated vesicles is mediated by a dual interaction of ␤-arrestin with both clathrin heavy chain and the ␤ 2 subunit of the heterotetrameric ...

Phospho-dependent binding of the clathrin AP2 adaptor complex to GABA A receptors regulates the efficacy of inhibitory synaptic transmission

“…In contrast, the GABA A R ␤-subunit 2 binding motif associates directly with a core domain in 2 (residues 283-394). Interestingly the arginine and lysine rich GABA A R ␤-subunit 2 (AP2) binding motif has significant similarity in amino acid content to an atypical AP2 binding motif recently identified in several other receptors and membrane proteins, including Syt 1 (16), the ␣1b adrenergic receptor (29) and AMPA receptors (30), supporting a conserved mechanism for AP2 binding in neuronal membrane proteins and neurotransmitter receptors.…”

Constitutive Signaling of the Human Cytomegalovirus-encoded Receptor UL33 Differs from That of Its Rat Cytomegalovirus Homolog R33 by Promiscuous Activation of G Proteins of the Gq, Gi, and Gs Classes

“…The contribution of both G␣ q/11 as well as G␤␥ subunits of the G i/o family in the UL33-mediated accumulation of InsP production was further corroborated by use of the kinasedeficient GRK2-K220R mutant (27). This mutant has been reported to scavenge both G␣ q/11 via interaction with its RGS domain and G␤␥ subunits via binding to its pleckstrin homology domain (27). As anticipated, co-expression of the kinasedeficient GRK2K220R mutant resulted in a complete inhibition of UL33-mediated InsP production (n ϭ 3; Fig.…”

Mutation of Three Residues in the Third Intracellular Loop of the Dopamine D2 Receptor Creates an Internalization-defective Receptor

“…Nonetheless, in the case of D 2 -A3, the precise conformation and interactions between these components may not be sufficient for internalization. Furthermore, despite the proximity between D 2 -A3 and ␤2-adaptin, D 2 -A3 may not be able to directly interact with other subunits of AP2 (42,43) or with another protein or lipid required for wild type receptor internalization. The inability of D 2 -A3 to internalize may also reflect characteristics of the dynamic interaction among GPCR, arrestin, and AP2 that are not captured in these BRET assays.…”