Identification of PAC1 receptor isoform mRNAs by real-time PCR in rat suprachiasmatic nucleus.
The pituitary adenylate cyclase-activating polypeptide (PACAP) has been implicated in the photic resetting of the rodent circadian clock in the suprachiasmatic nucleus (SCN). PACAP can exert its effects via VPAC1, VPAC2 and PAC1 G-protein coupled receptors. PAC1 and VPAC2, but not VPAC1, mRNA is expressed in rat SCN. A variety of PAC1 receptor splice variants have been described showing differences in ligand binding affinity and selectivity, G-protein coupling and ability to activate signal transduction pathways. The present experiments used PCR with isoform specific primers to determine which PAC1 variants are expressed in rat SCN. The PAC1(null) isoform and a variant containing a single 28-amino acid insert in the third intracellular (IC3) loop (hop1/2) were detected. No other IC3 variants (hip, hip-hop), N-terminal variants (PAC1(short), PAC1(very short) and PAC1(3a)) or the variant differing in transmembrane II and IV (PAC1TM4) were detected in SCN obtained at any time of day. A quantitative real-time PCR assay was established which measured combined expression of the PAC1(null/hop) variants in rat SCN during a 12:12-h light:dark (L:D) cycle. There was no significant variation of PAC1 mRNA expression (PAC1(null)+PAC1(hop)) with time of day. Nor was there a significant difference in the proportion of these two variants with time of day. These results indicate that the phase-dependency of the actions of PACAP on SCN firing and circadian behaviour are not mediated by changes in the level of expression of PAC1 receptor mRNA, nor by phase-dependent expression of PAC1 receptor variants with altered ligand binding, G-protein coupling or signalling characteristics.
Ajpru
Supaporn
S
Endocrinology and Reproduction Research Group, GKT School of Biomedical Sciences, Kings College London, London SE1 1UL, UK.
McArthur
Angela J
AJ
Piggins
Hugh D
HD
Sugden
David
D
D017398:Alternative Splicing / Q000235:genetics*; D000818:Animals; D002940:Circadian Rhythm / Q000235:genetics*; D005786:Gene Expression Regulation / Q000502:physiology; D008297:Male; D009479:Neuropeptides / Q000378:metabolism*; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D020033:Protein Isoforms / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone / Q000235:genetics*; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013493:Suprachiasmatic Nucleus / Q000378:metabolism*; D013997:Time Factors
C495403:Adcyap1 protein, rat; C495505:Adcyap1r1 protein, rat; D009479:Neuropeptides; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D020033:Protein Isoforms; D012333:RNA, Messenger; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D011979:Receptors, Pituitary Hormone; D051239:Receptors, Vasoactive Intestinal Peptide, Type II; D051238:Receptors, Vasoactive Intestinal Polypeptide, Type I; C495508:Vipr1 protein, rat; C495511:Vipr2 protein, rat