IDENTIFICATION OF THE CRITICAL DOMAINS OF THE DELTA-OPIOID RECEPTOR INVOLVED IN G-PROTEIN COUPLING USING SITE-SPECIFIC SYNTHETIC PEPTIDES

A large body of evidence implicates the second and third intracellular loops and the carboxyl-terminal portion of many G protein-coupled rec eptors as sites responsible for the interaction to G proteins. We synt hesized a number of peptides from selected sites of the murine delta-o pioid receptor and measured their ability to modify ligand-stimulated G protein activation and H-3 agonist binding to the receptor. In membr anes from Rat-1 fibroblasts transfected to express the murine delta-op ioid receptor stably (clone D2 cells), the delta-opioid agonist [D-Ser (2)-Leu(5)-Thr(6)]enkephalin (DSLET) stimulated high affinity GTPase a ctivity, which was inhibited by peptides that are derived from the pro ximal (i3.1) and the distal portions (i3.3) of the third intracellular loop with IC50 values of 15 +/- 5 and 50 +/- 4 mu M, respectively. Pe ptides i3.1 and i3.3 inhibited DSLET-stimulated [S-35]guanosine 5'-O-t hiotriphosphate binding in the same membranes. However, a peptide desi gnated i4, which was derived from a juxtamembranous region of the carb oxyl-terminal tail of the delta-opioid receptor, failed to alter agoni st-mediated high affinity GTPase activity or agonist-driven [S-35]guan osine 5'-O-thiotriphosphate binding. Specific binding of [H-3]DSLET to membrane preparations from clone D2 was reduced by peptides i3.1 and i4. Combinations of these peptides abolished detectable [H-3]DSLET bin ding in the same membranes. Peptides i3.1 and i3.3 also destabilized t he high affinity state of the receptor as assessed in H-3 agonist bind ing on membranes from neuroblastoma X glioma (NG108-15) hybrid cells, which express the delta-opioid receptor endogenously; furthermore, del ta-opioid receptor-stimulated GTPase activity in the same membranes wa s inhibited by peptides i3.1 and i3.3 but i4 was inactive. In contrast , peptides derived from the second intracellular loop (i2.1 and i2.2), an intermediate portion of the third intracellular loop (i3.2), and t he extreme amino-terminal region of the receptor were without effect i n these assays. These observations indicate that although peptides i3. 1, i3.3, and i4 act via different mechanisms, they provide evidence th at at least two sites of the third intracellular loop and part of the carboxyl-terminal tail of the delta-opioid receptor are important in t he interaction between this receptor and cellular G proteins. Collecti vely, these results provide novel information about regions of the del ta-opioid receptor that are involved in G protein coupling and high af finity agonist binding.