W276 mutation in the endothelin receptor subtype B impairs Gq coupling but not Gi or Go coupling

The mutation of W276 to cysteine within the human endothelin receptor subtype B (ET(B)R) is associated with Hirschsprung's disease, a congenital intestinal disease. The sequence surrounding W276 is highly conserved between the endothelin receptor subtypes A and B. We have introduced sets of mutations into W275 and W276 of the ET(B)R gene, and the corresponding W257 and W258 of the ET(A)R gene, and studied their coupling properties with G(i), G(o), and G(q) in reconstituted phospholipid vesicles. The prepared mutants all showed a similar affinity for endothelin-1. The W276C/ET(B)R and W276A/ET(B)R mutants had reduced activities in G(q) coupling but not in G(i)/G(o) coupling, while the W275A/ET(B)R displayed reduced activities in G(i)/G(q) coupling, with normal G(o) coupling. On the other hand, W257A/ET(A)R and W258A/ET(A)R exhibited wild-type activities in all examined G protein couplings. These results suggest that the defects in the G(q) signaling pathway by the ET(B)R are connected with Hirschsprung's disease and that the two conserved tryptophans play distinct roles in signal transduction by the two receptor subtypes. In addition, W275 and W276, which are thought to be located near the extracellular side of the transmembrane helix 5, play important roles in forming the active structure of ET(B)R.     

Interruption of endothelin signaling modifies membrane type 1 matrix metalloproteinase activity during ischemia and reperfusion

The matrix metalloproteinases (MMPs), in particular, membrane type 1 MMP (MT1-MMP), are increased in the context of myocardial ischemia and reperfusion (I/R) and likely contribute to myocardial dysfunction. One potential upstream induction mechanism for MT1-MMP is endothelin (ET) release and subsequent protein kinase C (PKC) activation. Modulation of ET and PKC signaling with respect to MT1-MMP activity with I/R has yet to be explored. Accordingly, this study examined in vivo MT1-MMP activation during I/R following modification of ET signaling and PKC activation. With the use of a novel fluorogenic microdialysis system, myocardial interstitial MT1-MMP activity was measured in pigs (30 kg; n = 9) during I/R (90 min I/120 min R). Local ET(A) receptor antagonism (BQ-123, 1 microM) and PKC inhibition (chelerythrine, 1 microM) were performed in parallel microdialysis probes. MT1-MMP activity was increased during I/R by 122 +/- 10% (P < 0.05) and was unchanged from baseline with ET antagonism and/or PKC inhibition. Selective PKC isoform induction occurred such that PKC-betaII increased by 198 +/- 31% (P < 0.05). MT1-MMP phosphothreonine, a putative PKC phosphorylation site, was increased by 121 +/- 8% (P < 0.05) in the I/R region. These studies demonstrate for the first time that increased interstitial MT1-MMP activity during I/R is a result of the ET/PKC pathway and may be due to enhanced phosphorylation of MT1-MMP. These findings identify multiple potential targets for modulating a local proteolytic pathway operative during I/R.     

Novel alternatively spliced exon in the extracellular ligand-binding domain of the pituitary adenylate cyclase-activating polypeptide (PACAP) type 1 receptor (PAC1R) selectively increases ligand affinity and alters signal transduction coupling during spermatogenesis

The expression of the paracrine signaling hormone pituitary adenylate cyclase-activating polypeptide (PACAP) is regulated in a cyclical fashion during the 12-day spermatogenic cycle of the adult rat testis. The precise functions of PACAP in the development of germ cells are uncertain, but cycle- and stage-specific expression may augment cAMP-regulated gene expression in germ cells and associated Sertoli cells. Here we report the existence of a heretofore unrecognized exon in the extracellular domain of the PACAP type 1 receptor (PAC1R) that is alternatively spliced during the spermatogenic cycle in the rat testis. This splice variant encodes a full-length receptor with the insertion of an additional 72 base pairs encoding 24 amino acids (exon 3a) between coding exons 3 and 4. The PAC1R(3a) mRNA is preferentially detected in seminiferous tubules and is expressed at the highest levels in round spermatids and Sertoli cells. Analyses of ligand binding and signaling functions in stably transfected HEK293 cells expressing the two receptor isoforms reveals a 6-fold increase in the affinity of the PAC1R(3a) to bind PACAP-38, and alterations in its coupling to both cAMP and inositol phosphate signaling pathways relative to the wild type PAC1R. These findings suggest that the extracellular region between coding exons 3 and 6 of PAC1R may play an important role in the regulation of the relative ligand affinities and the relative coupling to G(s) (cAMP) and G(q) (inositol phosphates) signal transduction pathways during spermatogenesis.