A Library of 7TM Receptor C-terminal Tails

Abstract

Adaptor and scaffolding proteins determine the cellular targeting, the spatial, and thereby the functional association of G protein-coupled seven-transmembrane receptors with co-receptors, transducers, and downstream effectors and the adaptors determine post-signaling events such as receptor sequestration through interactions, mainly with the C-terminal intracellular tails of the receptors. A library of tails from 59 representative members of the super family of seven-transmembrane receptors was probed as glutathione S-transferase fusion proteins for interactions with four different adaptor proteins previously proposed to be involved in postendocytotic sorting of receptors. Of the two proteins suggested to target receptors for recycling to the cell membrane, which is the route believed to be taken by a majority of receptors, ERM (ezrin-radixin-moesin)-binding phosphoprotein 50 (EBP50) bound only a single receptor tail, i.e. the ␤ 2 -adrenergic receptor, whereas N-ethylmaleimide-sensitive factor bound 11 of the tailfusion proteins. Of the two proteins proposed to target receptors for lysosomal degradation, sorting nexin 1 (SNX1) bound 10 and the C-terminal domain of G proteincoupled receptor-associated sorting protein bound 23 of the 59 tail proteins. Surface plasmon resonance analysis of the binding kinetics of selected hits from the glutathione S-transferase pull-down experiments, i.e. the tails of the virally encoded receptor US28 and the ␦-opioid receptor, confirmed the expected nanomolar affinities for interaction with SNX1. Truncations of the NK 1 receptor revealed that an extended binding epitope is responsible for the interaction with both SNX1 and G protein-coupled receptor-associated sorting protein as well as with Nethylmaleimide-sensitive factor. It is concluded that the tail library provides useful information on the general importance of certain adaptor proteins, for example, in this case, ruling out EBP50 as being a broad spectrumrecycling adaptor.Interaction of receptors with adaptor and scaffolding proteins is important for their biogenesis, their cellular sorting and targeting to the cell membrane, and their function at the membrane in complex with transducer molecules and downstream effector molecules as well as the subsequent internalization and post-endocytotic sorting of the receptors (1, 2). These interactions among receptors, adaptors, and scaffolding proteins are highly regulated processes that can be controlled by phosphorylation events (3), expression of receptor activating, or inactivating variants of adaptor proteins (4), by competition among adaptor proteins, and by competition between adaptor proteins and effector molecules (5). For the large family of G protein-coupled seven-transmembrane segment receptors (7TM 1 receptors), this field is still in its infancy and only a rather sketchy picture has emerged of relative importance of specific adaptor and scaffolding proteins for the biogenesis, function, and desensitization of these receptors. Methods such as yeast two-hybrid screening...

G Protein–Coupled Receptor Sorting to Endosomes and Lysosomes

“…GASP is a large 1395-amino acid cytoplasmic protein that lacks any known protein-protein interaction domains but contains 22 stretches of 15 acidic residues in the N-terminal region and a conserved C-terminal domain that is found in 10 related genes (96). GASP binds to many GPCRs, including those that internalize and efficiently recycle and/or sort through an ubiquitin-dependent lysosomal degradation pathway, such as the β 2 AR (95)(96)(97). Interestingly, a recent study indicates that GASP binds to a conserved region within the putative eighth α-helix of the DOR and β 1 AR (96), but how GASP functions precisely to regulate GPCR sorting within the endosomal-lysosomal system is not known (see Reference 38a for further discussion).…”

Dopamine D3 Receptors Are Down-regulated following Heterologous Endocytosis by a Specific Interaction with G Protein-coupled Receptor-associated Sorting Protein-1

“…Cells were incubated with M1-conjugated Alexa Fluor 647 to specifically label the mature receptors at the plasma membrane. Cells were then either left untreated or stimulated with agonist (10,20,45, and 90 min) and then were washed with PBS/EDTA to remove any remaining surface staining. Data are expressed as a percentage of the untreated and stripped samples where an increase in fluorescence is indicative of endocytosis, because internalized receptors are protected from the PBS/EDTA strip.…”

“…GASP-1 binds to distinct GPCRs from several other families as well, including the ␦-opioid receptor (6), the CB1 receptor (7), the bradykinin B 2 receptor (8), and the virally encoded chemokine receptor US28 (9), all of which are targeted for degradation after endocytosis. Members of these families that do not bind GASP-1, as well as many other GPCRs that do not bind GASP (10), are recycled rather than degraded after endocytosis. Post-endocytic degradation of GPCRs by GASP-1 has been shown to have behavioral relevance in vivo.…”

Interactions with PDZ Domain Proteins PIST/GOPC and PDZK1 Regulate Intracellular Sorting of the Somatostatin Receptor Subtype 5

“…Several interactions have been mapped to the membrane proximal domain of the C terminus, including the so-called helix 8, which encompasses the palmitoylation motifs found in most receptors of the rhodopsin-related type I family (3)(4)(5). These contacts especially involve proteins that act in the postendocytic, intracellular sorting of multiple receptors, i.e.…”