Among the two mammalian isoforms of cGMP-dependent protein kinase, cGKIα/β and cGKII, only the type II enzyme is expressed in epithelial cells and is equipped with an N-terminal myristyl anchor serving to facilitate its interaction with epithelial transporters, including the cystic fibrosis-gene encoded chloride channel CFTR [1], the TRPV5/6 renal and intestinal apical calcium channel [2], and the apical sodium/proton exchanger NHE3 (see below). Cyclic GMP activation of intestinal electrolyte and water secretion is triggered by a family of small, heat-stable peptides, the guanylins, and their microbial counterparts, the diarrheogenic heat-stable E. coli enterotoxins (STa). STa- and guanylin-activation of intestinal Cl^- secretion is severely impaired in cGKII-/- mice [3] and is completely lost in CFTR-/- and guanylyl cyclase C -/- mice, demonstrating the importance of a GC-C/cGKII signaling complex in the phosphorylation and activation of the CFTR-Cl^- channel in the luminal membrane. Such a paracrine/luminocrine mode of guanylin-induced CFTR activation is not limited to the enterocyte but is likely to operate also in other CFTR-expressing epithelia, including the pancreas, the parotid and submandibular glands, and Clara cells in the distal airways [4].

Our studies in the small intestine of cGKII-null mice have shown that the STa/cGMP/cGKII pathway, as part of its diarrheal action, in parallel inhibits NaCl absorption, most plausibly by inhibiting the major Na⁺ importer, NHE3. To unravel the molecular mechanism involved, adenoviral transfer of full-length cGKII, a myristoylation mutant (G2A-cGKII), and a cGKIβ-cGKII chimer was carried out in NHE3-transfected PS120 fibroblasts and its effect on cGMP-inhibition of NHE3 was examined. Inhibition appeared critically dependent on the co-expression of cGKII and the tandem-PDZ domain protein E3KARP (=NHERF2) which was not replaceable by the homologous protein NHERF1; in contrast cAMP/cAK inhibition was observed with either NHERF1 or NHERF2. Chimera studies showed that cGMP inhibition of NHE3 required the first 29 aminoacids of cGKII, which includes the myristoylation site, and that cGKI could inhibit NHE3 if anchored to the membrane. In pulldown, overlay and plasmon resonance assays, NHERF2 showed a low-affinity interaction with both cGKII and cGKIα/β through its PDZ2-C-terminal domain, which is also involved in NHE3 interaction. These findings suggest that NHERF2 functions as a "dual specificity" G kinase anchoring protein (GKAP) and that cGKII needs to be anchored at two sites (to the plasma membrane by myristyl-anchoring and to E3KARP) for inhibition of NHE3. Future studies in NHERF1-and NHERF2-knockout mice are aimed to determine the physiological importance of this mode of NHE3 regulation and its possible relevance for other transporters, including CFTR.