Cloning, expression, and in situ localization of rat intestinal cGMP-dependent protein kinase II.

Abstract

The cDNA for a membrane-associated cGMP-dependent protein kinase (cGK II) was cloned from rat intestine using reverse trnscriptase PCR and oligonucleotide primers encoding two conserved motifs of known cGMPdependent protein kinases and subsequently by screening a rat intestine cDNA library. A full-length clone encodes a protein of 761 amino acids with an estimated size of 87 kDa. Sequences of eight peptides from purified pig intestinal mucosa cGK II were found in the derived amino acid sequence of this done, identifying it as rat intestinal cGK II. Phylogenetic analysis showed that rat intestinal cGK II is less related to mammalian cGK I than to the Drosophila DG1 gene product and most closely related to a recently cloned mouse brain CGKH isoform. Like several other cGK sequences, that of cGK II contained a leucine/isoleucine heptad repeat motif that has been implicated in dimer formation in cGK I. Expression of cGK II cDNA in HEK 293 cells followed by subcellular fractionation revealed cGK H loalization in the cell particulate fraction, consistent with the membrane association of endogenous rat cGK II. On Northern blots, the major cGK H poly(A) RNA form was 4.8 kb, with minor forms of6.2 and 3.1 kb. The cGK H RNA was highly expressed in rat intestinal mucosa and was 20 times less abundant in rat brain and kidney. The localization of endogenous cGK II RNA in rat small intestine was shown by in situ hybridization to be in villous epithelial cells and to some extent in crypt cells.

Endogenous Type II cGMP-dependent Protein Kinase Exists as a Dimer in Membranes and Can Be Functionally Distinguished from the Type I Isoforms

“…However, recombinant mouse brain cGK II was reported to be soluble and dimeric after expression in mammalian and insect cells (5,16). In contrast, recombinant rat intestinal cGK II was found to be tightly bound to the membrane after expression in similar cell systems (4,17,18), but its oligomerization state was not established. Furthermore, the K a values for cGMP and cGMP analogs of recombinant rat intestine cGK II purified from Sf9 cells (17) differed considerably from those of recombinant mouse brain cGK II, which contained an additional N-terminal histidine tag (16).…”

“…The proteins in the second peak were identified by protein sequencing as C-terminal, proteolytic fragments of cGK II. The Nterminal sequences VPLDV and PPEF obtained from the 75-and the 70-kDa form, respectively, matched those of a 75-kDa cGK II fragment beginning at Val 101 and of a 70-kDa fragment starting at Pro 139 (4).…”

“…Sequence comparison and biochemical analysis revealed a large degree of similarity in the structural organization of cGK I and II (2)(3)(4)(5). Both isotypes possess two cGMP binding domains on one polypeptide chain which is covalently coupled to a catalytic domain.…”

cGMP Stimulation of Cystic Fibrosis Transmembrane Conductance Regulator Cl− Channels Co-expressed with cGMP-dependent Protein Kinase Type II but Not Type Iβ

“…Type II cGK is expressed predominantly in epithelial cells of the intestine (5-7), although it was also detected in kidney (6,8,9) and brain (6,8,10). In contrast, type I cGK, consisting of ␣ and ␤ isoforms, and shown to act as a key regulator of cardiovascular homeostasis (11,12), is not expressed in enterocytes (7).…”

“…Localization studies have suggested a key role for a recently cloned isotype of cGMP-dependent protein kinase (cGK), designated type II, as the mediator of the cGMP-provoked intestinal Cl Ϫ secretion (5)(6)(7). Type II cGK is expressed predominantly in epithelial cells of the intestine (5-7), although it was also detected in kidney (6,8,9) and brain (6,8,10).…”