Alternative Splicing in the N-terminal Extracellular Domain of the Pituitary Adenylate Cyclase-activating Polypeptide (PACAP) Receptor Modulates Receptor Selectivity and Relative Potencies of PACAP-27 and PACAP-38 in Phospholipase C Activation

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP)-27 and PACAP-38 are neuropeptides of the vasoactive intestinal peptide/secretin/glucagon family. We previously described alternative splicing of the region encoding the third intracellular loop of the PACAP receptor generating six isoforms with differential signal transduction properties (Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P. H., and Journot, L. (1993) Nature 365, 170 -175). In addition, we demonstrated that the potencies of the two forms of PACAP are similar for adenylate cyclase stimulation, whereas PACAP-38 is more potent than PACAP-27 in phospholipase C activation. In the present work, we document the existence of a new splice variant of the PACAP receptor that was characterized by a 21-aminoacid deletion in the N-terminal extracellular domain. We demonstrate that this domain modulates receptor selectivity with respect to PACAP-27 and -38 binding and controls the relative potencies of the two agonists in phospholipase C stimulation.Pituitary adenylate cyclase-activating polypeptides (PACAP) 1 are recently purified neuropeptides (1, 2) that are named according to their amino acid number, PACAP-27 and PACAP-38. The 27-amino acid form corresponds to the 27 N-terminal amino acids of PACAP-38 and shares 68% identity with vasoactive intestinal peptide (VIP). Two classes of PACAP binding sites were pharmacologically defined. Type I PACAP receptors bind PACAP-27 and -38 about two orders of magnitude more efficiently than VIP, whereas type II PACAP receptors do not discriminate between PACAP-27, -38, and VIP. At present, three PACAP/VIP receptor genes have been identified: PACAP 1 -R corresponds to PACAP type I receptors whereas PACAP/VIP 1 -R and PACAP/VIP 2 -R correspond to type II receptors. No VIP-specific receptor has so far been cloned.PACAP 1 -R is abundantly expressed in the brain, the pituitary and pineal glands, and the adrenal medulla. It mediates the neurotrophic action of PACAP-38 on PC12 cells (3) and sympathetic neuroblasts (4, 5). In the pituitary gland, PACAP-38 modulates the release of several hormones (1) and of interleukin-6 (6) through activation of PACAP 1 -R but also of PACAP/VIP 1 -R and PACAP/VIP 2 -R (7-11). PACAP 1 -R activation also controls proliferation of chromaffin cells (12) as well as catecholamine release by the adrenal medulla (13, 14).We and others (15-19) recently cloned the rat PACAP 1 -R cDNA. In addition, we demonstrated that PACAP 1 -R hnRNA is alternatively spliced and gives rise to six variants (18,20). Two cassettes named "hip" and "hop" are possibly inserted at the end of the third intracellular loop of the receptor. The resulting variants were named PACAP 1 -R s (the shortest form, without cassette), PACAP 1 -R hip, PACAP 1 -R hop1, PACAP 1 -R hop2, PACAP 1 -R hip-hop1, and PACAP 1 -R hip-hop2. They display differential signal transduction properties upon expression into LLC PK1 cells and Xenopus oocytes. The short as well as the hop variants potently activate both AC an...

Molecular Approximation between a Residue in the Amino-terminal Region of Calcitonin and the Third Extracellular Loop of the Class B G Protein-coupled Calcitonin Receptor

“…3) as did the Bpa 16 and Bpa 26 probes that we used previously, demonstrating its ability to be used in exploration of the ligand-binding sites using the photoaffinity-labeling approach. As expected, this intrinsic photoaffinity-labeling approach identified a residue (Leu 368 ) within the third extracellular loop domain of the CT receptor as the site of covalent attachment to the Bpa 8 probe, a region distinct from the amino-terminal domain of the CT receptor that was labeled by the Bpa 16 , Bpa 19 , and Bpa 26 probes (20,21).…”

“…It is worth of mentioning that the carboxyl-terminal residue Bpa 26 of CT ligand labeled a residue within the distal amino terminus of the receptor (20), the mid-region residues Bpa 16 (20) and Bpa 19 (21) labeled receptor residues within the amino terminus near the first transmembrane domain, and the amino-terminal residue Bpa 8 labeled a residue within the third extracellular loop of the receptor (the current work). These data suggest that when docking, the natural CT ligand is sited between two major docking domains (i.e.…”

Importance of the Amino Terminus in Secretin Family G Protein-coupled Receptors

“…In fact this domain has been shown to be critical for ligand binding by analysis of calcitonin-glucagon (9, 10) and calcitonin-PTH (8) receptor chimeras. The importance of the amino-terminal domain in ligand binding has been consistent for other members in the class B G protein-coupled receptor family, including receptors for secretin (22, 38 -41), vasoactive intestinal polypeptide (VIP) (38,40), PTH (42), and pituitary adenylate cyclase-activating polypeptide (43,44). This is also the domain labeled in analogous photoaffinity labeling studies for mapping of the binding domains of the secretin receptor (11, 12, 14 -17) (for review, see Ref.…”

Deconvolution of complex G protein–coupled receptor signaling in live cells using dynamic mass redistribution measurements

“…To effectively deliver cDNA coding for dominant-negative G13 (G13Q226L,D294N) into GPR55-HEK cells an electroporation method was used described previously 47 . DMR measurements were performed 48 h after transfection.…”