Cloning and characterization of the signal transduction of four splice variants of the human pituitary adenylate cyclase activating polypeptide receptor. Evidence for dual coupling to adenylate cyclase and phospholipase C

Abstract

Alternative splicing of two exons of the rat pituitary adenylate cyclase activating polypeptide (PACAP) receptor gene generates four major splice variants that are differentially expressed in specific tissues and variably coupled to intracellular second messengers. To evaluate the potential implications of these findings in human physiology, the human PACAP receptor gene was cloned. Alternative splicing about two exons of the gene allowed for four major splice variants that were subsequently identified on cDNA cloning. Each of the four splice variant cDNAs (null, SV-1, SV-2, and SV-3) was stably expressed in NIH/3T3 cells at similar receptor densities. For each splice variant, PACAP (both PACAP-38 and PACAP-27) had similar affinity and potency for stimulating either adenylate cyclase or phospholipase C. However, each receptor splice variant differed in their ligand-stimulated maximal response (efficacy) for total inositol phosphate accumulation with the SV-2 showing the greatest efficacy, followed by the null, SV-1, and SV-3 splice variants. Therefore, unlike the rat, PACAP binds and stimulates signal transduction with nearly equal affinity and potency for each of the receptor splice variants although with varying efficacy for the stimulation of phospholipase C. These results suggest a novel and potentially important mechanism for a single hormone to not only couple to dual signal transduction cascades but also elicit tissue-specific differential activation of phospholipase C in humans.

Fig. 1.. Amino acid model and partial gene structure of the hPACAP-R.

A, model of the predicted amino acid sequence for the hPACAP-R showing seven transmembrane-spanning regions (boxed amino acids) (22), potential sites for N-linked glycosylation (tridents) signal peptide cleavage site (arrow) (23), seven highly conserved cysteine residues (triangles), and the non-homologous rPACAP-R amino acids (letters outside the circles) (5). Amino acid products of the alternatively spliced exons, SV-1 and SV-2, are shown in brackets with their location in the third intracellular loop indicated by the arrow. B, model of the partial gene structure of the hPACAP-R gene showing the consensus sequences for the intron-exon splice sites for the exons SV-1 and SV-2. Two consecutive splice sites for the SV-2 exon (a and b) give rise to two possible splice variants (SV-2a and SV-2b).

Fig. 2.. Displacement of ¹²⁵I-PACAP-27 binding to NIH/3T3 cells stably expressing hPACAP-R splice variants.

NIH/3T3 cells stably expressing the indicated hPACAP-R splice variant were incubated with ¹²⁵I-PACAP-27 (50 pM) either alone or with the indicated concentrations of PACAP-38 or PACAP-27. Data are presented as percent of saturable binding in the absence of unlabeled ligand. Each value represents the mean ± standard error from at least five experiments performed in duplicate.

Fig. 3.. Ability of PACAP to increase intracellular cAMP in NIH/3T3 cells stably expressing hPACAP-R splice variants.

NIH/3T3 cells stably expressing the indicated hPACAP-R splice variant cDNA were incubated either alone or in the presence of increasing concentrations of PACAP-38 and PACAP-27. Data are shown as percent of maximal stimulation (maximally stimulated cAMP minus nonstimulated cAMP) and represent the means of three experiments performed in triplicate.

Fig. 4.. Ability of PACAP to increase total inositol phosphates in NIH/3T3 cells stably expressing hPACAP-R splice variants.

NIH/3T3 cells stably expressing the indicated hPACAP-R splice variant cDNA were incubated either alone or in the presence of increasing concentrations of PACAP-38 and PACAP-27. Data are shown as percent of maximal stimulation (maximally stimulated IP minus nonstimulated IP) and represent the means of three experiments performed in triplicate.

Fig. 5.. Determination of the efficacy of the PACAP stimulated increase in cAMP for each of the hPACAP-R splice variants stably expressed in NIH/3T3 cells.

Clones of each of the hPACAP-R splice variants expressing similar receptor densities were incubated either alone or in the presence of increasing concentrations of PACAP-38. Data are presented as the increase in [³H]cAMP in cpm/10⁵ cells over basal (in the absence of added peptide) and represent the means of three experiments performed in triplicate. Similar results were observed for PACAP-27 (data not shown).

Fig. 6.. Determination of the efficacy of the PACAP stimulated increase in total inositol phosphates for each of the hPACAP-R splice variants stably expressed in NIH/3T3 cells.

Clones of each of the hPACAP-R splice variants expressing similar receptor densities were incubated either alone or in the presence of increasing concentrations of PACAP-38. Data are presented as the increase in total [³H]inositol phosphates in cpm/10⁵ cells over basal (in the absence of added peptide) and represent the means of three experiments performed in triplicate. Similar results were observed for PACAP-27 (data not shown).