We have used an expression-cloning strategy to isolate a cDNA encoding a somatostatin (somatotropin release-inhibiting factor, SRIF) receptor from rat cortex and hippocampus. A positive clone was identified by autoradlography after binding of radiolabeled SRIF to COS-1 cells previously transfected with pools of cDNA clones. The deduced amino acid sequence of the receptor displays sequence and structural homology to the family of G-protein-coupled receptors. The affinity of various SRIF analogs to the expressed receptor resembles their effects on growth hormone release from pitnitary cells. In addition, the dbution of the mRNA in various tissues corresponds to that described for native SRIF receptors. Therefore, we conclude that we have isolated a rat brain SRIF receptor cDNA.Somatostatin (somatotropin release-inhibiting factor, SRIF), a neuropeptide initially isolated from the hypothalamus, was shown to act on many different cell types by inhibiting the secretion of hormones, including growth hormone (GH), insulin, glucagon, gastrin, and secretin (1-3). In addition, SRIF was shown to act as a neurotransmitter (4-6). Therefore, SRIF has widespread functions as a modulator of neuronal activity as well as endocrine and exocrine secretion. These regulatory effects are mediated by specific membrane receptors on the SRIF target tissues (7). High-affinity, saturable binding sites have been demonstrated in cortex, hippocampus, substantia nigra, pituitary gland, pancreas, adrenal cortex, and several tumor types-for instance, endocrine, breast, lung, or brain tumors (8)(9)(10)(11). Radiolabeled SRIF analogs have been used extensively to characterize the binding properties of SRIF receptors on the various target tissues. Previously, it has been shown that SRIF receptors are coupled to GTP-binding proteins (G proteins) (12)(13)(14)(15) In the present study we describe the isolation and primary structure of a SRIF receptor cDNA, the pharmacology of the expressed receptor, and the distribution of the mRNA.* The approach chosen for cloning of the receptor cDNA included the construction of a cDNA expression library with RNA isolated from the developing rat cortex and hippocampus, transfection of pools of clones into COS-1 cells, and identification of cells expressing SRIF receptors by autoradiography after binding of iodinated ligands (21,22). MATERIALS AND METHODScDNA Cloing. RNA was purified from cortex and hippocampus dissected from brains of 6-day-old rats by the guanidinium thiocyanate/acid phenol method (23). Poly(A)+ RNA was enriched by two subsequent passages over an oligo(dT)-cellulose column as described (24). Five micrograms of poly(A)+ RNA was used in the synthesis of oligo(dT)-primed double-stranded cDNA using the Amersham cDNA synthesis system. After addition ofphosphorylated Dra III adapters (pGTC GAC CAC CTC and pGTG GTC GAC) the cDNAs were size-selected by gel filtration [Sepharose CL-2B (Pharmacia), 39 x 1.5 cm column; running buffer = 1 M KCI/35 mM Tris'HCl, pH 8.3/30 mM KH2PO4/1 mM EDTA; flow rate,...

A DNA segment homologous to the third exons of the serotonin 1C and 2 receptor genes was isolated from a mouse genomic library. The positions of the introns flanking these exons were conserved in the three genes. To examine whether the new fragment was part of an active gene, we used a quantitative PCR protocol to analyse rat RNAs from different tissues and ages. The gene was expressed in stomach fundus at an abundance of 1 × 10(5) mRNA molecules. This tissue contracts in response to serotonin via a receptor that has previously resisted classification. We constructed a cDNA library from rat stomach fundus and isolated clones containing 2020 bp inserts with open reading frames of 465 amino acids comprising seven putative membrane‐spanning regions. The protein was transiently expressed in COS cells and binding of serotonergic ligands to the membranes was analysed. The pharmacological profile resembled that described for the serotonin‐stimulated contraction of the stomach fundus. After expression of this receptor in Xenopus oocytes, the application of serotonin triggered the typical chloride current which presumably results from the activation of phospholipase C. The coupling to this response system was less efficient than that of the 5‐HT1C or 5‐HT2 receptors.

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A cDNA of a novel form of type V adenylyl cyclase has been cloned from rabtut myocardmm using ohgonucleotlde probes denved from peptides that were produced by enzymatic cleavage of punfied heart cyclase A correspondmg mRNA (6 kb) has been detected m rabtnt myocarchal tissue by Northern blot analysis The cDNA encodes a protein of 1,264 ammo acids exhlbltmg 12 putatwe membrane-spanning regons m its hydroplnhclty profile Sequence comparison to two other previously published type V adenylyl cyclases reveals ammo-terminal domams of different length and low correlative homology, whereas the rest of the sequences IS almost identical The nonconserved ammo-terminal region of the subtype consists of 214 ammo acids and exceeds the length of the others by 40 and 80 residues, respectively Its presence m membrane preparations from different tissues has been confirmed immunologically usmg an antibody directed against a synthetic peptide The cloned adenylyl cyclase was functionally expressed m COS-1 cells to attam an enzymatrc actlvlty 3 S-to 1Cfold above control m the presence of forskohn

The gene mutation in the mouse, ‘arrested development of rightingresponse’, adr, causes a defect of chloride conductance of the muscle fibre membrane leading to the symptoms of myotonia [Mehrke, G., Brinkmeier, H. and Jockusch, H. (1988) Muscle & Nerve 11, 440‐446]. In fast muscle, the myotonic phenotype is accompanied by a drastic reduction of the Ca2+‐binding protein, parvalbumin. Messenger RNA levels in organs of myotonic (ADR) mice were analysed. In fast muscles of the mutant, in‐vitro‐translatable parvalbumin mRNA was strongly reduced, whereas the mRNA for the slow‐muscle‐specific protein, p19/6.8, was increased. In contrast, the parvalbumin mRNA in thecerebellum was not affected by the adr mutation. A reduction of the two parvalbumin mRNA species (700 and 1100 nucleotides) in ADR fast muscle and unaltered prvalbumin cDNA as a probe. The mRNA level for another Ca2+‐binding protein, calmodulin, was low in muscle and high in the central nervous system but was unaffected by the mutation. When adr/adrmice were fed a diet containing the membrane‐stabilising drug, tocainide, the levels inmuscle of the mRNAs for parvalbumin and p19/6.8 were partially normalised.