While trying to identify new members of the somatostatin receptor family of G protein-coupled receptors, we isolated cDNAs from a mouse brain library encoding two related receptor-like proteins, designated msl-l and msl-2, of 380 and 372 amino acids, respectively. There was 61% identity and 71% similarity between the sequences of msl-1 and msl-2.Among members of the G protein-coupled receptor superfamily, the sequences of both msl-1 and msl-1 were most closely related to those of the somatostatin receptors (SSTRs), having -35% identity with the sequence of SSTR1. Transient expression in COS-1 cells showed that msl-1 and msl-2 did not bind somatostatin. Rather they bound opioids selectively and with high affnity and had the pharmacological properties of K and 8 opioid receptors, respectively. Indeed, the sequence of msl-2 was identical to that of a 8 opioid receptor recently doned by other workers. Functional characterization of K/msl-l and 8/msl-2 opioid receptors showed that they were coupled to G proteins and mediated opioid receptor class-specific agonist inhibition of forskolin-stimulated cAMP formation. RNA blotting studies and in situ hybridization histochemistry showed that K opioid receptor mRNA was expressed at high levels in brain in the neocortex, hippocampus, amygdala, medial habenula, hypothalamus (arcuate and paraventricular nuclei), locus ceruleus, and parabrachial nucleus, suggesting that this receptor may play a role in arousal and regulation ofautonomic and neuroendocrine functions.

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Opium and its derivatives are potent analgesics that can also induce severe side effects, including respiratory depression and addiction. Opioids exert their diverse physiological effects through specific membrane-bound receptors. Three major types of opioid receptors have been described, termed 8, c, and ,t Opioids such as morphine are used clinically for the management of pain (1). However, the use of opioids has undesirable side effects-including respiratory depression, decreased gastrointestinal motility, sedation, nausea, and mood changes. Other major limitations include abuse potential, tolerance, and physical dependence. Morphine and the endogenous opioid peptides, the enkephalins, endorphins, and dynorphins, exert their physiological effects through membrane-bound receptors expressed in the central and peripheral nervous systems and in target tissues.The three major types ofopioid receptors, 8, K, and ,u, have recently been cloned and functionally characterized (2-5). They belong to the Asp-Arg-Tyr (DRY)-containing subfamily of seven transmembrane-spanning receptors. There is =60% amino acid identity among the sequences of the 8, K, and , opioid receptors. The sequences of the putative membranespanning segments (TM I-VII) and the three intracellular loops connecting these segments are highly conserved, whereas the sequences of the extracellular NH2-termini segments, second and third extracellular loops, and the intracellular COOH termini are divergent. It seems reasonable to assume that these divergent extracellular regions may be responsible for the distinct ligand-binding profiles ofthe 8, K, and 1i receptors. To test this hypothesis, we have exchanged the extracellular NH2 termini of the mouse 8 and K receptors (3, 4) and examined the abilities of these chimeric receptors to bind 8-and K-selective agonists and antagonists, as well as to mediate inhibition of adenylyl cyclase activity. METHODSGeneration of Chimeras. To exchange NH2 termini between the mouse 8 and K opioid receptors, a common restriction site, Spe I, was generated at an equivalent position in the cDNAs in the region encoding the first trainsmembrane domain without altering the amino acid sequence of either receptor. Site-directed mutagenesis was done by using the Altered Sites in vitro mutagenesis system (Promega) and 27-mer oligonucleotides containing the Spe I site (8-receptor oligonucleotide, CTGGGCAACGTACTAGTCATGTTTG-GC, and K-receptor oligonucleotide, GTGGGCAATTCAC-TAGTCATGTTTGTC). After digestion with Spe I and the appropriate 5' and/or 3' enzymes, the cDNA fragments encoding the NH2 and COOH termini of 8 and K were isolated from a 1.2% low-melting-point agarose gel. Fragments encoding the NH2 terminus of 8 receptor and the COOH terminus of K receptor and vice versa were ligated together and cloned into the mammalian expression vector pCMV-6c. Truncated 8 and K receptors were generated by ligating the fragments encoding the COOH termini directly into the expression vector. Translation of the receptor sequences in these ...

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Somatostatin (SRIF) is a neurotransmitter that produces its multiple effects in the CNS through interactions with membrane-bound receptors. Subtypes of SRIF receptors are found in the CNS that are distinguished by their sensitivities to the cyclic hexapeptide MK-678, such that SRIF1 receptors are sensitive to MK-678 and SRIF2 receptors are insensitive to MK-678. In the present study, we further examined the selectivities of a series of structurally diverse SRIF analogues for SRIF receptor subtypes. SRIF receptors were labeled by 125I-Tyr11-SRIF, which has indistinguishable affinities for SRIF receptor subtypes. The inhibition by MK-678 was incomplete, indicating this peptide is highly selective for a subtype of SRIF receptor that we have termed the SRIF1 receptor. The binding of 125I-MK-678 to SRIF1 receptors was monophasically inhibited by SRIF, the octapeptides (such as SMS-201-995), and the hexapeptides (such as MK-678), consistent with the highly selective labeling of a subtype of SRIF receptor. In contrast, the smaller CGP-23996-like analogues did not inhibit 125I-MK-678 binding to SRIF1 receptors. The binding of 125I-CGP-23996 to SRIF receptors was inhibited by SRIF and the octapeptides with Hill coefficients of less than 1, indicating that 125I-CGP-23996 labels multiple SRIF receptor subtypes. The hexapeptides and CGP-23996-like compounds produced only partial inhibitions of 125I-CGP-23996 binding, which were additive, indicating selective interactions of these compounds with the different receptor subpopulations labeled by 125I-CGP-23996. 125I-Tyr11-SRIF binding and 125I-CGP-23996 binding to SRIF receptors were likewise only partially affected by 100 microM guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), a concentration that completely abolishes specific 125I-MK-678 binding to SRIF1 receptors. The component of 125I-CGP-23996 labeling that was sensitive to GTP gamma S was also MK-678 sensitive. Thus, two subpopulations of SRIF receptors exist in the CNS. The SRIF1 receptor is sensitive to cyclic hexapeptides such as MK-678 and to GTP gamma S but insensitive to smaller CGP-23996-like compounds. The SRIF2 receptor is sensitive to the CGP-23996-like compounds and can be selectively labeled by 125I-CGP-23996 in the presence of high concentrations of the hexapeptides or GTP gamma S because, unlike the SRIF1 receptor, the SRIF2 receptor is insensitive to these agents. The SRIF receptor subtype-selective peptide analogues will be useful in the future characterization of the functions mediated by SRIF receptor subtypes in the CNS.

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