ABSTRACT,u opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described 8 and K opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.

Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently-cloned p, S and K receptors. XORlS, a rat brain cDNA whose predicted translation product displays 67-72% homology with those encoded by ~1, 61 and ~1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XORlL variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XORl mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate hgands nor reproducible opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.

Cellular responsiveness to the inhibitory peptide somatostatin (SRIF) or its clinically used analogs can desensitize with agonist exposure. While desensitization of other seven-transmembrane domain receptors is mediated by receptor phosphorylation and/or internalization, the mechanisms mediating SRIF receptor (sst) desensitization are unknown. Therefore, we investigated the susceptibility of the sst2A receptor isotype to ligandinduced desensitization, internalization, and phosphorylation in GH-R2 cells, a clone of pituitary tumor cells overexpressing this receptor. A 30-min exposure of cells to either SRIF or the analog SMS 201-995 (SMS) reduced both the potency and efficacy of agonist inhibition of adenylyl cyclase. Internalization of receptor-bound ligand was rapid (t1 ⁄2 ‫؍‬ 4 min) and temperature-dependent. SRIF and SMS increased the phosphorylation of the 71-kDa sst2A protein 25-fold within 15 min. Receptor phosphorylation was dependent on both the concentration and time of agonist exposure and was not affected by pertussis toxin pretreatment, indicating that receptor occupancy rather than second messenger formation was required. Receptor phosphorylation was also stimulated by phorbol 12-myristate 13-acetate activation of protein kinase C. Both ligand-stimulated and phorbol 12-myristate 13-acetate-stimulated receptor phosphorylation occurred primarily on serine. These studies are the first demonstration of agonist-dependent desensitization, internalization, and phosphorylation of the sst2A receptor and suggest that phosphorylation may mediate the homologous and heterologous regulation of this receptor.The somatostatin peptides 1 influence endocrine, exocrine, and neuronal function through binding to a family of six G protein-coupled receptors (sst1, sst2A, sst2B, sst3, sst4, and sst5) (1, 2). Within the SRIF receptor family, sst2A receptor mRNA has been detected in many tissues including the brain, pituitary, pancreas, spleen, small intestine, and stomach (1, 2), and the receptor protein has recently been shown to be widely distributed in the mammalian brain (3). Thus, this receptor isotype mediates many of the central and peripheral actions of SRIF.Early studies on the signal transduction mechanisms activated by SRIF showed that sst receptors elicited their actions predominantly via pertussis toxin-sensitive G proteins (1, 2, 4). Thus, SRIF inhibition of adenylyl cyclase and Ca 2ϩ channels, as well as SRIF stimulation of K ϩ channels, phospholipase C, serine/threonine and tyrosine phosphatases, arachidonic acid release, and mitogen-activated protein kinases are inhibited by pertussis toxin treatment (5-12). However, some actions of SRIF, such as stimulation of other tyrosine phosphatases as well as inhibition of Na/H exchange, are pertussis toxin-insensitive (13,14). The network of signaling pathways activated by individual sst receptor isotypes is largely unknown. Signaling mechanisms have been especially difficult to elucidate in the native environment of the receptors because most SRIF target cells exp...

Earlier work in Aplysia californica has indicated that sensitization of the gill reflex, a simple form of learning, is produced by cAMP-dependent protein phosphorylation which regulates the flux of ions in sensory neurons of the abdominal ganglion. These changes in ion flux result in the enhanced release of neurotransmitter from synapses of the sensory neurons which, in turn, mediate the behavior. Because it can be presumed that protein phosphorylation regulates the functioning of ion channel proteins, we have characterized cAMP-binding proteins photoaffinity labeled with 8-N3-cAMP and have found that, unlike other tissues, the nervous system contains a great variety of binding species. Also unlike other tissues, several of the binding proteins in neurons are associated with membrane, and these components are concentrated in fractions enriched in nerve endings. Selectivity of phosphorylation, not only between substrates in cytosol and membrane but also between different regions of the cell, is thus possible because of the variety of cAMP-binding proteins in neurons. We think that these membrane-associated binding proteins are the most likely candidates for the regulatory subunits of the cAMP-dependent kinases that control the functioning of ion channel proteins at the synapse.