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.

The nonselective human corticotropinreleasing factor receptor 1 (hCRF-R1) and the ligand-selective Xenopus CRF-R1 (xCRF-R1) were compared. To understand the interactions of sauvagine and ovine CRF, both highaffinity ligands for hCRF-R1 but surprisingly weak ligands for xCRF-R1, chimeric receptors of hCRF-R1 and xCRF- When only two of these three amino acids were mutated, no effect on the ligand selectivity was observed. On the basis of these data, it is suggested that amino acids 70-89 of CRF-R1 are important for the ligand binding site.

Abstract:The aim of the present study was to identify the N-terminal regions of human corticotropin-releasing factor (CRF) receptor type 1 (hCRF-R1) that are crucial for ligand binding. Mutant receptors were constructed by replacing specific residues in hCRF-R1 with amino acids from the corresponding position in the N-terminal region of the human vasoactive intestinal peptide receptor type 2 (hVIP-R2). In cyclic AMP stimulation and CRF binding assays, it was established that two regions within the N-terminal domain were crucial for the binding of CRF receptor agonists and antagonists: one region mapping to amino acids 43-50 and a second amino acid sequence extending from position 76 to 84 of hCRF-R1. Recently, it was found that the latter sequence plays a very important role in determining the high ligand selectivity of the Xenopus CRF-R1 (xCRF-R1). Replacement of amino acids 76 -84 of hCRF-R1 with residues from the same segment of the hVIP-R2 N terminus markedly reduced the binding affinity of CRF ligands. Mutation of Arg 76 or Asn 81 but not Gly 83 of hCRF-R1 to the corresponding amino acids of xCRF-R1 or hVIP-R2 resulted in 100 -1,000-fold lower affinities for human/rat CRF, rat urocortin, and astressin. These data underline the importance of the N-terminal domain of CRF-R1 in high-affinity ligand binding. Key Words: Corticotropin-releasing factor receptor 1 mutagenesis-Ligand binding-Cyclic AMP-Corticotropinreleasing factor binding site. J. Neurochem. 72, 388 -395 (1999).The 41-amino acid peptide corticotropin-releasing factor (CRF) (for references, see Vale et al., 1981) is the main integrator of the stress response (Dunn and Berridge, 1990;Owens and Nemeroff, 1991). Two major subtypes of the CRF receptor (CRF-R) have been identified in vertebrates as belonging to the superfamily of G protein-coupled receptors (GPCRs): CRF-R type 1 (CRF-R1) and type 2 (CRF-R2). The cDNAs for CRF-R1 and CRF-R2 have been cloned from human Vita et al., 1993;Liaw et al., 1996), rat (Chang et al., 1993;Perrin et al., 1993;Lovenberg et al., 1995), mouse (Vita et al., 1993;Kishimoto et al., 1995;Perrin et al., 1995;Stenzel et al., 1995), chicken (Yu et al., 1996), and amphibian (Dautzenberg et al., 1997) tissues. Two functional splice variants, CRF-R2␣ and CRF-R2␤, have been found for CRF-R2. CRF-R2␣ cDNA encodes a protein of 411-413 amino acids (Lovenberg et al., 1995;Liaw et al., 1996;Dautzenberg et al., 1997), whereas the CRF-R2␤ protein comprises 430 -438 amino acids (Kishimoto et al., 1995;Lovenberg et al., 1995;Perrin et al., 1995;Stenzel et al., 1995;Valdenaire et al., 1997). Both splice variants differ in the N-terminal region and are ϳ70% identical to CRF-R1.High-affinity ligand binding is an important prerequisite for the signal transduction of GPCRs. Small nonpeptidic molecules can bind to the transmembrane domains (TMs) of their receptors (Dixon et al., 1987;Wheatley et al., 1988;Strader et al., 1989), whereas the contribution of extracellular domains (ECs) and TMs has been identified as parts of the binding site of GP...

Abstract:The nonselective human corticotropin-releasing factor (hCRF) receptor 1 (hCRFR1) and the ligandselective Xenopus CRFR1 (xCRFR1), xCRFR2, and hCRFR2␣ were compared. To understand the interactions of hCRF, ovine CRF (oCRF), rat urocortin (rUcn), and sauvagine, ligands with different affinities for type 1 and type 2 CRFRs, chimeric and mutant receptors of hCRFR1, xCRFR1, hCRFR2␣, and xCRFR2 were constructed. In cyclic AMP stimulation and CRF-binding assays, it was established that different extracellular regions of CRFR1 and CRFR2 conferred their ligand selectivities. The ligand selectivity of xCRFR1 resided in five N-terminal amino acids, whereas the N-terminus of both CRFR2 proteins did not contribute to their ligand selectivities. Chimeric receptors in which the first extracellular domain of hCRFR1 replaced that of hCRFR2␣ or xCRFR2 showed a similar pharmacological profile to the two parental CRFR2 molecules. Chimeric receptors carrying the N-terminal domain of xCRFR1 linked to hCRFR2␣ or xCRFR2 displayed a novel pharmacological profile. hCRF, rUcn, and sauvagine were bound with high affinity, whereas oCRF was bound with low affinity. Furthermore, when three or five residues of xCRFR1 ( , Gly 81 , Val 83 ) were introduced into receptor chimeras carrying the N-terminus of hCRFR1 linked to xCRFR2, the same novel pharmacology was observed. These data indicate a compensation mechanism of two differentially selecting regions located in different domains of both xCRFR1 and CRFR2.

From brain, heart and muscle tissue of the tree shrew (Tupaia belangeri), a higher order mammal, cDNA clones were isolated that encoded two functional splice variants of the corticotropin-releasing factor (CRF) type 2 receptor (CRF-R2). The first, full-length splice variant, amplified from brain and heart tissue, encoded a CRF receptor protein that is 410 amino acids in length and approximately 96% homologous to human CRF-R2alpha. The second, full-length splice variant, derived from skeletal muscle tissue, encoded a 437-amino acid CRF receptor protein that is approximately 92% homologous to human CRF-R2beta. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) amplifications and RNase protection analyses, showed that tree shrew CRF-R2alpha (tCRF-R2alpha) and tree shrew CRF-R2beta (tCRF-R2beta) were coexpressed in brain tissue but not in heart and skeletal muscle tissue. Finally, human embryonic kidney 293 (HEK293) cells stably transfected with tCRF-R2alpha and tCRF-R2beta were used to demonstrate that the CRF analogs urocortin and sauvagine bind with significantly greater affinity (21- to 140-fold) to these two CRF-R2 splice variants than do human/rat and ovine CRF analogs. In keeping with these results of our CRF binding studies, EC50 values were substantially lower for urocortin-and sauvagine-stimulated than for h/rCRF-and oCRF-stimulated cyclic AMP accumulation in HEK293 cells stably transfected with tCRF-R2alpha or tCRF-R2beta cDNAs. The tree shrew therefore constitutes an important animal model in which to investigate the role of CRF receptor subtypes in the stress response.