NHERF family and NHE3 regulation

Abstract

The intestinal and renal proximal tubule brush border (BB) Na+-H+ exchanger NHE3 binds to members of the NHERF (Na+-H+ exchanger regulatory factor) family. These are four proteins (current most used names include NHERF1, NHERF2, PDZK1 and IKEPP) which are related to each other, are present in locations in or close to the BB, and scaffold a variable series of proteins in NHE3-containing complexes in a dynamic manner that is altered by changes in signal transduction which affects NHE3 activity. The specific roles of these proteins in terms of NHE3 regulation as well as interactions with each other and with their many other substrates are only now being defined. Specificity for only one member of the NHERF family in one example of NHE3 regulation, inhibition by elevation in cGMP, is used to describe how NHERF family proteins are involved in NHE3 complex formation and its regulation. In this case, NHERF2 directly binds cGKII in the brush border to form an NHE3 complex, with cGKII also associating with the BB via its myristoylation.

Figure 1. NHERF Family

A, localization of PDZ domain-containing proteins to epithelial cell brush border, basolateral membranes, tight junctions, Golgi or endosomes. B, NHERF gene family: shown are the suggested names (NHERF 1–4), multiple previous names of the proteins, protein–protein interacting domains, and number of amino acids. ERM, ezrin, radixin, moesin.

Figure 2. Evolution of the PDZ domains of the mammalian NHERF family from C. elegans and D. melanogaster

PDZ domains of the NHERF family were defined using ScanSite MotifScan. The 12 human NHERF family PDZ domains were individually analysed for sequence relationship by the MegAlign program (DynaStar, Inc.). The domains were grouped by closest relationships. Relationship with C. elegans and D. melanogaster proteins were also analysed by MegAlign. All protein names and sequences were obtained from NCB1. C. elegans has two PDZ domain-containing proteins that are related to the NHERF family. CO1F6.6 contains one PDZ domain while 4J893 contains two PDZ domains, one identical to that in CO1F6.6 and the other 5% identical. This suggests that the second PDZ domain of 4J893 may have evolved by gene duplication. Each of the two worm PDZ domains is a precursor to certain groups of PDZ domains of the human NHERF family. The D. melanogaster proteins CG10939-PA and CG32758-PA each contain one PDZ domain (10% identical to each other) and are precursors to the human NHERF family.

Figure 3. NHE3 is present in large, multiprotein complexes in the membranes of multiple cell types, including the ileal Na⁺ absorptive cell brush border

NHE3 expressed in multiple cell types was solubilized in 1% Triton X-100 and placed on 5–35% discontinuous sucrose density gradients and separated by size, as described previously (Poulat et al. 1997). In cells expressing the largest amount of NHE3 on the plasma membrane (Caco-2 cell) and when the plasma membrane NHE3 was compared to total NHE3, there were generally larger NHE3 complexes on the plasma membrane (Li et al. 2004).

Figure 4. NHE3 complexes in the ileal brush border are dynamic, increasing in size with regulation by carbachol/Ca²⁺ elevation

Sucrose density gradient separation similar to that in Fig. 3 (Poulat et al. 1997) was carried out with ileal Na⁺ absorptive cell BB prepared after 10 min exposure of ileum to carbachol in vitro. Western analysis demonstrated that the size of NHE3 complexes were larger after carbachol exposure. Similar to the increase in size of NHE3 after carbachol addition, α-actinin-4 was distributed in multiple, large complexes which increased in size after carbachol, in parallel with the changes in size of NHE3 complexes (Li et al. 2004).

Figure 5. Model of cGMP inhibition of NHE3 in OK cell brush border and PS120 cells

NHERF2 is required for the cGMP inhibition of NHE3. NHERF2 acts as a cGMP kinase anchoring protein (GKAP), forming a complex of NHE3, NHERF2 and cGKII. cGKII is required for cGMP to inhibit NHE3. To carry out this effect, cGKII must be fixed in two locations, by its N-terminus via myristoylation to the plasma membrane and to the second PDZ domain of NHERF2 (Cha et al. 2005). Given the BB localization of guanylate cyclase C, cGMP is generated at the BB where it probably acts on the cGKII, which is part of the BB NHE3 complex.