Molecular cloning and expression of a pituitary somatostatin receptor with preferential affinity for somatostatin-28.
Using the polymerase chain reaction technique with degenerative primers, we obtained from a rat pituitary cDNA library a cDNA fragment, rAP236, that exhibited considerable homology to known receptors that belong to the guanine nucleotide-binding protein (G protein)-coupled receptor superfamily. Oligonucleotides to this fragment were used as probes to obtain a full-length cDNA from the rat pituitary cDNA library. This clone, rAP6-26, encoded a 383-amino acid protein with seven putative transmembrane domains that are characteristic of G protein-coupled receptors. The predicted amino acid sequence of the rAP6-26 cDNA exhibits 56-66% homology to recently cloned somatostatin (SRIF) receptors. Membranes prepared from COS-7 cells transfected with the rAP6-26 cDNA showed specific binding of 125I-Tyr11-SRIF, thus identifying the cDNA clone as a novel SRIF receptor. Radioligand binding competition analysis using somatostatin-28 (SRIF-28) and a number of cyclic SRIF analogs revealed that SRIF-28 was the most potent competitor of 125I-Tyr11-SRIF binding, with a approximately 30-fold greater affinity for the receptor than that of SRIF. In addition, binding of 125I-Tyr11-SRIF was markedly reduced in the presence of Na+ ions and GTP, indicating coupling of rAP6-26 receptors to inhibitory G proteins in COS-7 membranes. In adenylyl cyclase assays, forskolin-induced cAMP accumulation was inhibited by SRIF and SRIF-28, thus confirming that the rAP6-26 cDNA encodes a functional receptor protein. By Northern blot analysis, a approximately 2.6 kilobase mRNA encoding the receptor was present in the pituitary but not in the liver, small intestine, kidney, pancreas, cerebellum, or cortex. Lack of receptor mRNA expression in the brain was confirmed by in situ hybridization histochemical studies. Thus, we report the cloning of a novel rat pituitary SRIF receptor, termed SSTR4, that has marked preferential affinity for SRIF-28 and is linked to inhibition of adenylyl cyclase.
Laboratory of Cell Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
A M
AM
O'Carroll
S J
SJ
Lolait
M
M
König
L C
LC
Mahan
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D004247:DNA; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017481:Receptors, Somatostatin / Q000235:genetics* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013004:Somatostatin / Q000378:metabolism*; D053561:Somatostatin-28; D013379:Substrate Specificity
D011498:Protein Precursors; D012333:RNA, Messenger; D017481:Receptors, Somatostatin; D013004:Somatostatin; D053561:Somatostatin-28; D004247:DNA