Truncation of the A1 Adenosine Receptor Reveals Distinct Roles of the Membrane-proximal Carboxyl Terminus in Receptor Folding and G Protein Coupling

Abstract

The carboxyl terminus (C-tail) of G protein-coupled receptors is divergent in length and structure and may represent an individualized cytoplasmic domain. By progressively truncating the A 1 adenosine receptor, a G i/o -coupled receptor with short cytoplasmic stretches, we identify two inherent functions of the C-tail, namely a role in receptor export from the endoplasmic reticulum (ER) and a role in G protein coupling. Deletion of the last 22 and 26 amino acids (of 36) reduced and completely abolished surface expression of the receptor, respectively. The severely truncated receptors were retained in the ER and failed to bind ligands. If overexpressed, even a substantial portion of the fulllength receptor was retained in the ER in a form that was not functional. These data indicate that folding is rate limiting in export from the ER and that the proximal segment of the carboxyl terminus provides a docking site for the machinery involved in folding and quality control. In addition, the proximal portion is also important in G protein coupling. This latter role was unmasked when the distal portion of the C-tail (the extreme 18 amino acids, including a palmitoylated cysteine) had been removed; the resulting receptor was functional and transferred the agonist-mediated signal more efficiently than the full-length receptor. Signaling was enhanced because the coupling affinity increased (by 3-fold), which translated into a higher agonist potency. Thus, the distal portion of the carboxyl terminus provides for an autoinhibitory restraint, presumably by folding back and preventing G protein access to the proximal part of the C-tail.The cytoplasmic face of a G protein-coupled receptor (GPCR) executes the signal transfer reaction through interaction with G proteins and accessory non-GTP-binding protein components. The interface is composed of three intracellular loops that connect the trans-membrane helices and a fourth loop created by a segment of 8 -16 amino acids adjacent to transmembrane helix 7 (TM7), which is tethered to the phospholipid bilayer by one or two palmitoylated cysteine residues. In individual instances, each of the intracellular segments has been shown to contribute to G protein coupling and signaling (1). The second intracellular loop (i 2 ), which contains the conserved D(E)RY(W) motif and the third intracellular loop (i 3 ), in particular its amino-and carboxyl-terminal ends, are key elements. Between receptor types and even among related subtypes, sequence similarities in the cytoplasmic domains are scarce; this variability suggests that, among the cytoplasmic segments, some are reserved for receptor functions more divergent than G protein coupling.The receptor carboxyl terminus varies considerably in length ranging from nil (in the GnRH receptor) to more than 200 amino acids (in the Ca 2ϩ /Mg 2ϩ -sensing receptor). Distinct functional properties have been assigned to individual carboxyl termini. In rhodopsin and the metabotropic glutamate receptor (subtypes IV, VI, and VII), the proximal portion repre...

A Membrane-proximal, C-terminal α-Helix Is Required for Plasma Membrane Localization and Function of the G Protein-coupled Receptor (GPCR) TGR5

“…Similar findings, i.e. a reduced functionality and retention in the ER, have been demonstrated for other GPCRs with a truncated membrane-proximal, intracellular C terminus, such as the luteinizing hormone/chorionic gonadotropin receptor (21), the vasopression 2 receptor (22), and the A1 adenosine receptor (23). This indicates that the cellular signaling response is determined by the amount of functionally active receptor in the plasma membrane (PM) (24).…”

Role of the C-terminal di-leucine motif of 5-HT1A and 5-HT1B serotonin receptors in plasma membrane targeting

“…These studies showed that the short cytosolic C-terminal tail of both receptors plays a crucial role in their targeting. As also reported for other GPCRs (Bermak et al, 2001;Duvernay et al, 2004;Oksche et al, 1998;Pankevych et al, 2003;Rodriguez et al, 1992;Tai et al, 1999), this region appeared to be necessary for the transport of 5-HT 1A R and 5-HT 1B R to the cell surface, because truncated receptors without the C-terminal domain were sequestrated within the endoplasmic reticulum (ER) in LLC-PK1 cells. Furthermore, these studies also demonstrated that the cytosolic C-terminal tail of 5-HT 1B R contains an axonal-apical targeting signal (Jolimay et al, 2000).…”

Di-acidic Motifs in the Membrane-distal C Termini Modulate the Transport of Angiotensin II Receptors from the Endoplasmic Reticulum to the Cell Surface

“…The requirement of the membrane-proximal C-terminal portion for ER export has been described for a number of GPCRs, including AT1R, ␣ 2B -AR, rhodopsin, vasopressin V2 receptor, dopamine D1 receptor, adenosine A1 receptor, melanin-concentrating hormone receptor 1, and luteinizing hormone/choriogonadotropin receptor (24,53,54). Mutagenesis studies have also identified a number of highly conserved motifs in the membrane-proximal C termini essential for GPCR export from the ER, such as the E(x) 3 LL, FN(x) 2 LL(x) 3 L, and F(x) 3 F(x) 3 F motifs (22,26,27).…”