Galanin receptor 1 gene expression is regulated by cyclic AMP through a CREB-dependent mechanism.
The galanin receptor-1 (GalR1) protein belongs to a family of G protein-coupled receptors for the neuropeptide galanin (GalR1, GalR2 and GalR3) distributed throughout the central and peripheral nervous system. Activation of galanin receptors by their ligands results in increased feeding, impaired learning, enhanced opiate analgesia and decreased opiate place preference. We have shown that opiate withdrawal, which is known to increase levels of cAMP in the locus coeruleus (LC), results in an increase in the number of galanin binding sites and the level of GalR1 mRNA in the LC. We have isolated a 3.6-kb fragment 5' of the inititiation codon of the mouse GalR1 gene and generated a series of deletion mutations of this fragment driving expression of luciferase for use in transient transfection assays in PC12 and Cath.a cell lines. Treatment with forskolin, but not dideoxyforskolin, up-regulates GalR1 transcription, likely through elevation of cAMP levels. The region between - 1050 and - 700 base pairs upstream of exon one is necessary both for basal activity of the GalR1 promoter and for forskolin-mediated increases in transcription. The forskolin effect can be blocked by simultaneous mutation of a CRE-like site and a CRE/DRE-like site, but not mutation of either site alone. Gel shift and super-shift experiments demonstrate that the transcription factor CREB can bind to both sites and is likely to be responsible for the cAMP-mediated increase in GalR1 promoter activity. This study provides a molecular mechanism for the increased GalR1 expression in the LC seen following opiate withdrawal.
Zachariou
V
V
Department of Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06508, USA.
Georgescu
D
D
Kansal
L
L
Merriam
P
P
Picciotto
M R
MR
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D018387:Codon, Initiator / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020932:Nerve Growth Factor / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000187:drug effects; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide / Q000096:biosynthesis* / Q000235:genetics; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects; D012689:Sequence Homology, Nucleic Acid; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D018387:Codon, Initiator; D017362:Cyclic AMP Response Element-Binding Protein; D044088:Receptors, Galanin; D018013:Receptors, Neuropeptide; D005576:Colforsin; D020932:Nerve Growth Factor; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate