Cloning and functional characterization of a sigma receptor from rat brain.
We have cloned a sigma receptor from rat brain and established its functional identity using a heterologous expression system. The cloned cDNA (1,582 bp long) codes for a protein of 223 amino acids that possesses a single putative transmembrane domain. The amino acid sequence of the rat brain sigma receptor is highly homologous to that of the sigma receptor recently cloned from guinea pig liver and a human placental cell line but is not related to any other known mammalian receptors. When expressed in HeLa cells, the rat brain sigma receptor cDNA leads to a two- to threefold increase in haloperidol binding, and this cDNA-induced binding is sensitive to inhibition by several sigma receptor-specific ligands. Kinetic analysis using the heterologous expression system has revealed that the rat brain sigma receptor interacts with haloperidol with an apparent dissociation constant (K(D)) of 3 nM. Functional expression of the cloned rat brain sigma receptor in HeLa cells also leads to an increase in the binding of two other sigma ligands, namely, (+)-pentazocine and (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (PPP). Pharmacological characterization of the cloned rat brain sigma receptor reveals that it exhibits severalfold higher affinity for clorgyline than for 1 ,3-di(2-tolyl)guanidine, it interacts with progesterone and testosterone, and its interaction with PPP is markedly enhanced by phenytoin. In addition, transfection of MCF-7 cells, which do not express type 1 sigma receptor mRNA or activity, with the cloned rat brain cDNA leads to the appearance of haloperidol-sensitive binding of (+)-pentazocine, a selective type 1 sigma receptor ligand. These data show that the cloned rat brain cDNA codes for a functional type 1 sigma receptor. Northern blot analysis with poly(A)+ RNA isolated from various rat tissues has indicated that the sigma receptor-specific transcript, 1.6 kb in size, is expressed abundantly in liver and moderately in intestine, kidney, brain, and lung.
Seth
P
P
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912-2100, USA.
Fei
Y J
YJ
Li
H W
HW
Huang
W
W
Leibach
F H
FH
Ganapathy
V
V
D000595:Amino Acid Sequence; D000818:Animals; D000927:Anticonvulsants / Q000494:pharmacology; D000928:Antidepressive Agents / Q000494:pharmacology; D001483:Base Sequence; D001667:Binding, Competitive / Q000187:drug effects / Q000502:physiology; D001923:Brain Chemistry; D002822:Choriocarcinoma; D003001:Cloning, Molecular; D003010:Clorgyline / Q000494:pharmacology; D018076:DNA, Complementary; D018492:Dopamine Antagonists / Q000494:pharmacology; D015870:Gene Expression; D015723:Gene Library; D006146:Guanidines / Q000494:pharmacology; D006220:Haloperidol / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D008969:Molecular Sequence Data; D009294:Narcotics / Q000494:pharmacology; D010423:Pentazocine / Q000494:pharmacology; D010672:Phenytoin / Q000494:pharmacology; D010920:Placenta / Q000737:chemistry; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017480:Receptors, sigma / Q000145:classification* / Q000235:genetics*; D013256:Steroids / Q000494:pharmacology
D000927:Anticonvulsants; D000928:Antidepressive Agents; D018076:DNA, Complementary; D018492:Dopamine Antagonists; D006146:Guanidines; D009294:Narcotics; D012333:RNA, Messenger; D017480:Receptors, sigma; D013256:Steroids; D010672:Phenytoin; D006220:Haloperidol; C050232:1,3-ditolylguanidine; D003010:Clorgyline; D010423:Pentazocine