Opioid Receptor Types Selectively Cointernalize with G Protein-Coupled Receptor Kinases

Activation of G protein-coupled receptors (GPCRs) may bring about their disappearance from the cell membrane, and it is commonly accepted that G protein-coupled receptor kinases (GRKs) play a key function in this mechanism. Opioid receptors belong to the family of GPCRs and are substrates of GRKs. We examined the fate of and -opioid receptors and GRK2 and 3 in living cells during the process of receptor sequestration, using laser scanning microscopy. For visualization purposes, receptors and kinases were tagged at their respective C terminus with a fluorophore. The opioid receptors were fused to enhanced green fluorescence protein (EGFP), and the GRKs were linked to red fluorescence protein (DsRed). The cDNAs of these constructs served for transfection of human embryonic kidney 293 cells and neuroblastoma-glioma hybrid cells (NG 108-15), respectively. We report that activation of -opioidEGFP receptors triggers a rapid translocation of cytosolic GRKDsRed toward the cell membrane, which in turn releases vesicles carrying both green fluorescent -receptors and red fluorescent GRKs. Phosducin, a G scavenger, completely prevents translocation of GRKs and the formation of vesicles. In analogous experiments with -opioid receptors fused to EGFP, we observed that receptor activation also discharges green fluorescent vesicles. In contrast to -receptors, -receptors failed to trigger accumulation of GRK2 or 3 at the membrane, and no cointernalization of -opioid receptors with GRK2 or 3 was observed. The results suggest that -opioid receptors, but not -receptors, cointernalize with GRK2 or 3. G protein-coupled receptors (GPCRs) represent transmembrane proteins that mediate signals to the interior of cells. Activation of these receptors by extracellular ligands triggers their immediate phosphorylation, followed by uncoupling from intracellular G proteins and sequestration (Carman and Benovic, 1998). It is likely that the strength of receptor activation controls the intensity of receptor phosphorylation (Zhang et al., 1998) by enzymes termed G protein-coupled receptor kinases (GRKs). The GRKs consist of at least six related isoenzymes (GRK1–6), which transfer phosphate groups onto serine and threonine residues located close to the C terminus of receptor protein (Pitcher et al., 1998). GRKs mainly reside in the cytosol (Sterne-Marr and Benovic, 1995) and are suggested to translocate immediately upon GPCRactivation to the plasma membrane where they anchor at G subunits (Lefkowitz, 1993) to phosphorylate agonistoccupied receptors. Thereafter, GRKs are suggested to dissociate from G and redistribute to the cytosol. Arrestin will bind to the phosphorylated receptor to terminate signal transmission and to initiate receptor internalization (Carman and Benovic, 1998; Reiter et al., 2001). Opioid receptors are members of the GPCR family (Cox, 1993). Their activation triggers the dissociation of inhibitory acting Gi/o proteins, resulting in attenuation or even termination of transmembrane signaling, a process defined as receptor desensitization (Ferguson, 2001). However, agonistoccupied receptors may undergo endocytosis followed either by down-regulation or their recycling to the cell membrane (Carman and Benovic, 1998). These mechanisms are differently affected both by the individual intrinsic activities of agonists (Yabaluri and Medzihradsky, 1997; Kovoor et al., 1998) and by the composition of GRKs with which the cells are equipped (Ferguson, 2001). Notably, the opiate agonist morphine fails to induce -opioid receptor internalization but will gain the capacity to sequester receptors as the cytosolic concentration of GRK2 is increased (Roettger et al., 1997; Zhang et al., 1998). Furthermore, the multiplicity of opioid receptors on the one hand and the at least six GRKs on the other suggests distinct interactions between these entities. Indeed, -opioid receptors are controlled by GRK2 (Zhang et al., 1998; Ferguson, 2001), GRK3 (Carman and Benovic, 1998; Hurle, 2001), and even by GRK6 (Hurle, 2001). -Opioid receptors are phosphorylated by GRK2 (Schulz et al., 1998b; Harrison et al., 1998), GRK3 (Carman and Benovic, 1998), and GRK5 (Harrison et al., 1998). Additional information links the function of GRKs to chronic opioid effects, as an ABBREVIATIONS: GPCR, G protein-coupled receptor; DADLE, [D-Ala,D-Leu]-enkephalin; DAMGO, [D-Ala,N-Me-Phe,glycinol]-enkephalin; DPDPE, [D-Pen,D-Pen]-enkephalin; EGFP, enhanced green fluorescence protein; DsRed, red fluorescence protein; GRK, G protein-coupled receptor kinase; GTP S, guanosine-5 -O-(thio)triphosphate; HEK, human embryonic kidney; LSM, laser scanning microscopy; wt, wild-type. 0022-3565/02/3002-376–384$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 2 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4499/960584 JPET 300:376–384, 2002 Printed in U.S.A. 376 at A PE T Jornals on M ay 4, 2017 jpet.asjournals.org D ow nladed from increased abundance and activity of distinct GRKs was communicated for the state of opioid dependence and tolerance (Terwillinger et al., 1994; Kovoor et al., 1998; Ozaita et al., 1998; Hurle, 2001). Thus, our present knowledge favors the notion that GRKs account for the phosphorylation of activated opioid receptors, but there is no indication that the action of a distinct GRK is confined to a specific opioid receptor type. Although our knowledge regarding phosphorylation and internalization of GPCRs has been derived mainly from experiments with cell fragments, the present study was designed to explore more closely the interrelation between activated opioid receptors and certain GRKs during the process of receptor sequestration in living cells. For this reason opioid receptors were fused with enhanced green fluorescence protein (EGFP; Heim et al., 1995), and GRKs were linked to red fluorescence protein (DsRed; Wall et al., 2000). The fusion proteins were expressed in cell lines (HEK 293, NG 108-15) and confocal laser microscopy was applied to track the fate of green fluorescent opioid receptors and red fluorescent GRKs in life cells. The major outcome of this study is the demonstration that activated -opioid receptors and receptor kinases 2 and 3 cointernalize. In contrast, internalization of -opioid receptors was not found accompanied by any GRK under investigation. Materials and Methods Chemicals. The biochemical reagents were of analytical grade and purchased from Calbiochem (Deisenhofen, Germany) and Sigma (Taufkirchen, Germany). Opioid receptor ligands deltorphin II, [D-Ala,N-Me-Phe,glycinol]-enkephalin (DAMGO), [D-Pen,D-Pen]enkephalin (DPDPE), and [D-Ala,D-Leu]-enkephalin (DADLE) were from Bachem (Heidelberg, Germany), sufentanil was from Janssen Pharmaceuticals (Beerse, Belgium), and naloxone was from DuPont (Geneva, Switzerland). Guanosine-5 -O-(thio)-triphosphate (GTP S) was from Sigma. The Taq-DNA polymerase was purchased from New England Biolabs (Schwalbach, Germany), G418 sulfate was from Calbiochem (Bad Soden, Germany), and the restriction enzymes were purchased from MBI Fermentas (St. Leon-Rot, Germany). The enzyme inhibitor Complete came from Roche Molecular Biochemicals (Mannheim, Germany). Radio-Labeled Tracers. I-cAMP (2000 Ci/mmol) was obtained from Amersham (Braunschweig, Germany) and [H]diprenorphine (32 Ci/mmol) was purchased from PerkinElmer Life Sciences (Dreieich, Germany). Cell Culture Reagents. Chemicals were purchased from Invitrogen (Karlsruhe, Germany), fetal calf serum from PAN (Nürnberg, Germany), and cell culture material from NUNC GmbH & Co. KG (Wiesbaden, Germany). Antibodies. The anti-cAMP antiserum was from Bio-Yeda (Rehovot, Israel), the anti-DsRed antibody from CLONTECH (Heidelberg, Germany), and all other antibodies were delivered from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Cell Culture. HEK 293 cells and neuroblastoma x glioma 108–15 cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum as described (Ammer and Schulz, 1993). Experiments were conducted with cells at 60% con-