Distinct Subcellular Localization for Constitutive and Agonist-modulated Palmitoylation of the Human δ Opioid Receptor

Abstract

Protein palmitoylation is a reversible lipid modification that plays important roles for many proteins involved in signal transduction, but relatively little is known about the regulation of this modification and the cellular location where it occurs. We demonstrate that the human ␦ opioid receptor is palmitoylated at two distinct cellular locations in human embryonic kidney 293 cells and undergoes dynamic regulation at one of these sites. Although palmitoylation could be readily observed for the mature receptor (M r 55,000), [ 3 H]palmitate incorporation into the receptor precursor (M r 45,000) could be detected only following transport blockade with brefeldin A, nocodazole, and monensin, indicating that the modification occurs initially during or shortly after export from the endoplasmic reticulum. Blocking of palmitoylation with 2-bromopalmitate inhibited receptor cell surface expression, indicating that it is needed for efficient intracellular transport. However, cell surface biotinylation experiments showed that receptors can also be palmitoylated once they have reached the plasma membrane. At this location, palmitoylation is regulated in a receptor activation-dependent manner, as was indicated by the opioid agonist-promoted increase in the turnover of receptor-bound palmitate. This agonistmediated effect did not require receptor-G protein coupling and occurred at the cell surface without the need for internalization or recycling. The activation-dependent modulation of receptor palmitoylation may thus contribute to the regulation of receptor function at the plasma membrane.Palmitoylation, a post-translational lipid modification, plays an important role in the structure and function of a variety of proteins involved in cell signaling (1, 2). Among them several G protein-coupled receptors (GPCRs), 3 mainly belonging to family A, have been shown to be palmitoylated on one or more cysteine residues located in the proximal region of their C-terminal tail (2). The attached palmitate has therefore been proposed to promote the formation of a fourth cytoplasmic loop through its anchoring in the membrane (3). Consequently, palmitoylation may have a profound effect on the local conformation of this domain and possibly controls interaction of GPCRs with specific regulatory proteins. It has been shown to display diverse roles in GPCR function, including G protein coupling, desensitization, trafficking, and targeting of the receptors (2). Unlike other lipid modifications such as myristoylation and prenylation, palmitoylation is a highly dynamic modification. The 16-carbon saturated palmitate that is attached to the specific cysteine residues via an acyl-thioester bond has been demonstrated to turn over rapidly (4 -8) and palmitoylation/depalmitoylation cycles may therefore regulate protein function. It has been shown that the palmitoylation/depalmitoylation cycle is activation-dependent for several G protein ␣-subunits (4, 9 -11), endothelial nitric-oxide synthase (12), and several GPCRs (the ␤ 2 -adrenergic (5, 13), D 1...

Coexpression of δ- and μ-opioid receptors in nociceptive sensory neurons

“…These patterns are shown by immunostaining of both exogenously expressed Myc-DOR1 and endogenous DORs using antibodies against DOR1 3-17 or DOR1 [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] . Thus, these antibodies against different N-terminal regions of DOR1 might recognize DORs in different states of activation, conformation, glycosylation, and/or palmitoylation (41)(42)(43)(44). Moreover, the translocation of exogenously expressed Myc-DOR1 from LDCVs to the cell surface in peptidergic small DRG neurons of Tac1 −/− mice supports an essential role of the DOR/protachykinin interaction in sorting DORs into LDCVs (19).…”

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

“…Analysis of Cell Surface Receptor Expression by Flow Cytometry-The total pool of cell surface receptors was labeled with anti-c-Myc antibody (9E10, ascites fluid 1:500) or anti-HA antibody (HA-7, 1:500) and phycoerythrin (PE)-conjugated rat anti-mouse IgG 1 (BD Biosciences, 1 g/ml) as described earlier (15). Fluorescein-NTX was used to label the ligand binding competent pool of cell surface receptors (Table 2 and Quantitation of Receptor Internalization by Flow CytometryFor assessing constitutive internalization, cell surface receptors were labeled with anti-c-Myc antibody (9E10, 2 g/ml; Santa Cruz Biotechnology) in complete DMEM for 30 min at 37°C.…”

“…It has been used extensively as a model to investigate GPCR biosynthesis. During its transit from the ER to the plasma membrane, it undergoes extensive modifications, including N-and O-glycosylation and palmitoylation (14,15). Its maturation is, however, inefficient (14), and a large fraction of synthesized receptors is degraded in proteasomes (16).…”

Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8

“…The palmitoylation of proteins known to be modified early in the secretory pathway is unaffected by BFA treatment (30)(31)(32). Similarly, syntaxin 8 might be modified in the ER or Golgi.…”

“…In BFA-treated cells, there is a collapse of Golgi cisternae into a mixed ER-Golgi compartment and tubulation of the trans-Golgi network and endosomal membranes (28,29). Treatment of cells with BFA results in the retention of newly synthesized H-ras (30), the delta opioid receptor (31) and the viral hemagglutin proteins on intracellular membranes (32), but does not affect their palmitoylation. This suggests that the palmitoyltransferases that modify these proteins are present in the mixed ER/ Golgi compartment.…”