Three full-length cDNAs encoding functional splice variants of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1) were isolated from Y-79 retinoblastoma cells and human cerebellum. Although the third intracellular loops of the three splice variants were identical, their N-terminal extracellular domains differed. The first full-length PAC1 variant, PAC1normal (PAC1n), encoded the entire N-terminus, whereas the second variant named PAC1short (PAC1s) was deleted by 21 amino acids (residues 89-109). Finally, the third variant, named PAC1very short (PAC1vs), was deleted by 57 amino acids (residues 53-109). Using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis, it was established that all three variants were expressed in neuronal tissues. Binding- and cAMP studies using human embryonic kidney 293 (HEK293) cells stably transfected with PAC1n, PAC1s and PAC1vs showed significant differences in the affinities and selectivities towards PACAP38, PACAP27 and VIP. PAC1n bound PACAP38 and PACAP27 with affinities in the low nanomolar range whereas VIP was bound with up to 400-fold lower affinity. PAC1vs preferentially bound PACAP38 (Ki=121 nM) and PACAP27 (Ki=129 nM) over VIP (Ki>1000 nM) but with 100-fold lower affinity than PAC1n. Surprisingly, PAC1s unselectively bound all three ligands with high affinity. These data indicate that residues 53-88 within the N-terminal domain of the PAC1 are important for high affinity ligand binding, whereas residues 89-109 determine the receptor's ligand selectivity.

A cDNA clone encoding corticotropin-releasing factor (CRF) type 1 (CRF-R1) has been isolated from the tree shrew Tupaia belangeri with a PCR-based approach. The full-length cDNA encoded a 415-amino-acid protein with highest sequence identity (Ϸ98%) to human CRF-R1 and slightly less identity to rat or mouse CRF-R1 (Ϸ97%). Only eight amino acids (residues 3, 4, 6, 35, 36 and 39 in the Nterminus, residue 232 in transmembrane domain 4 and residue 410 in the C-terminus) differed between tree shrew CRF-R1 (tCRF-R1) and human CRF-R1 (hCRF-R1). tCRF-R1 mRNA was detected by semiquantitative RT-PCR and RNase protection analysis in the pituitary and in brain areas such as amygdala, brainstem, cerebellum, cortex, olfactory bulb, and striatum. In peripheral organs, only weak expression of tCRF-R1 mRNA was observed in ovary, testis, and adrenal gland. Binding studies using human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R1 showed that the CRF agonists ovine CRF (K D ϭ 1.28 nM), human/rat CRF (K D ϭ 1.09 nM), urocortin (K D ϭ 0.37 nM) and sauvagine (K D ϭ 0.77 nM), respectively, were bound with significantly higher affinities than the CRF antagonist astressin (K D ϭ 12.4 nM). In agreement with the binding data half maximum effective EC 50 values of 0.83 nM (human/rat CRF), 1.41 nM (ovine CRF), 1.25 nM (rat urocortin) and 0.71 nM (sauvagine) were calculated when the cAMP production in HEK293 cells stably transfected with tCRF-R1 was stimulated with the four CRF analogues. These data underline the close relationship between human and tree shrew CRF-R1.Keywords : receptor cloning; receptor expression ; ligand binding; cyclic AMP.Corticotropin-releasing factor (CRF), a 41-amino-acid peptide, was originally identified by its ability to regulate the secretion of adrenocorticotropic hormone from the anterior pituitary [1]. The peptide is the main regulator of the stress response [2,3]. Two CRF receptor (CRF-R) subtypes have been identified in vertebrate species; CRF receptor type 1 (CRF-R1) cDNA, encoding a polypeptide of 415Ϫ420 amino acids has been cloned from human, rat [6,7] [9]. The receptor exists in at least two different functional splice variants, named CRF-R2A (411Ϫ413 residues) and CRF-R2β (430Ϫ438 amino acids). Both splice variants differ in the N-terminal region and show an overall identity to vertebrate CRF-R1 of Ϸ70% [9Ϫ15].CRF-R1 mRNA was mainly found in brain and the anterior pituitary [4Ϫ6, 9, 16Ϫ18]. In peripheral organs, CRF-R1 is only weakly expressed [5, 19Ϫ21]. In contrast to CRF-R1, CRF-R2A mRNA is limited to discrete regions of the rodent brain including hypothalamus, lateral septum and hippocampus but is almost Fax: ϩ41 61 6884484. E-mail: frank.dautzenberg@roche.com Abbreviations. CRF, corticotropin releasing factor; CRF-R, CRF receptor; oCRF, ovine CRF ; h/rCRF, human/rat CRF ; rUcn, rat urocortin; 5′-RACE and 3′-RACE, rapid amplification of cDNA ends. not detectable in the pituitary [17,18]. CRF-R2β is mainly expressed in peripheral organs such as heart, lung and skeletal muscle an...

Recent reports suggest that oral choline supplement may alter the cerebral choline/creatine (Cho/Cr) ratio and might be used to treat neurodegenerative disorders of cholinergic transmission. Using both 1H and 31P MRS, we reexamined the Cho/Cr ratio and quantified cerebral choline and its major constituents: phosphoethanolamine (PE), phosphorylcholine (PC), glycerophosphorylethanolamine (GPE), and glycerophosphorylcholine (GPC). In the four brain locations examined, no significant increases in Cho/Cr, [Cho], or in its major constituents were found in response to an oral challenge of 50 mg/kg of choline bitartrate. Oral choline did not significantly affect human cerebral metabolism in the short term.