Using small-angle x-ray scattering, we have observed the cGMPinduced elongation of an active, cGMP-dependent, monomeric deletion mutant of cGMP-dependent protein kinase (⌬ 1-52 PKG-I␤). On saturation with cGMP, the radius of gyration of ⌬ 1-52 PKG-I␤ increases from 29.4 ؎ 0.1 Å to 40.1 ؎ 0.7 Å, and the maximum linear dimension increases from 90 Å ؎ 10% to 130 Å ؎ 10%. The elongation is due to a change in the interaction between structured regulatory (R) and catalytic (C) domains. A model of cGMP binding to ⌬ 1-52 PKG-I␤ indicates that elongation of ⌬ 1-52 PKG-I␤ requires binding of cGMP to the low-affinity binding site of the R domain. A comparison with cAMP-dependent protein kinase suggests that both elongation and activation require cGMP binding to both sites; cGMP binding to the low-affinity site therefore seems to be a necessary, but not sufficient, condition for both elongation and activation of ⌬ 1-52 PKG-I␤. We also predict that there is little or no cooperativity in cGMP binding to the two sites of ⌬ 1-52 PKG-I␤ under the conditions used here. Results obtained by using the ⌬ 1-52 PKG-I␤ monomer indicate that a previously observed elongation of PKG-I␣ is consistent with a pure change in the interaction between the R domain and the C domain, without alteration of the dimerization interaction. This study has revealed important features of molecular mechanisms in the biochemical network describing PKG-I␤ activation by cGMP, yielding new insight into ligand activation of cyclic nucleotide-dependent protein kinases, a class of regulatory proteins that is key to many cellular processes.C yclic guanosine monophosphate (cGMP) is a second messenger signaling molecule that is central to the regulation of many physiological processes, including smooth muscle tone, visual transduction, platelet aggregation, bone growth, and electrolyte and fluid homeostasis (see reviews in refs. 1-3). Its known targets are ion channels, which can alter cellular cation or anion transport; phosphodiesterases, which degrade cyclic nucleotides; and the cGMP-dependent protein kinases (PKGs), which are believed to be responsible for most intracellular cGMP actions. The PKG [first discovered by Kuo and Greengard (4)] is a serine͞threonine protein kinase whose substrates include ion channels and pumps, receptors, and enzymes that control intracellular Ca 2ϩ concentrations. In its active form, PKG can transfer the ␥-phosphate from ATP to target proteins, preferentially targeting serines in substrates having consensus sequence RRXSX, with some exceptions (1, 5). Phosphorylation of target proteins by PKG effects smooth muscle relaxation by reduction of cellular Ca 2ϩ . The PKGs and cAMP-dependent protein kinases (PKA) are homologous enzymes, and the two enzyme families share many similarities in biochemical function and domain organization.PKG is a dimer comprised of two identical monomers (1-3). Each monomer of PKG contains a regulatory domain (R) and a catalytic domain (C) on a single polypeptide chain. The monomers dimerize via interactions ...

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2-Arachidonoylglycerol is oxygenated by cyclooxygenase-2 to form prostaglandin glyceryl esters. Previous work in this laboratory has suggested that PGE2-G activates a novel G protein-coupled receptor in a murine macrophage-like cell line, RAW 264.7. To probe the structural determinants for the putative receptor in RAW 264.7 cells, a panel of 10 analogs was tested for their ability to increase intracellular calcium. These analogs included PGE2- and PGF2α– ethanolamide, 4 PGE2 glyceryl ester analogs, and 4 PGF2α glyceryl ester analogs. The glyceryl ester analogs differed in the positioning of the hydroxyl groups in the glycerol moiety and the type of linker (ester, amide, or thioester) of the prostaglandin to the glycerol moiety. Compounds were also evaluated in a human non-small cell lung cancer cell line (H1819). The glycerol moiety was required for the calcium response. All glyceryl ester analogs but not ethanolamides caused a concentration-dependent increase in calcium levels in both RAW 264.7 and H1819 cells. An amide or ester linkage was preferable to a thioester linkage. The EC50 values did not significantly change when the positioning of the hydroxyls was varied. This calcium response induced by the glyceryl ester analogs appears to be independent of the putative hydrolysis products, PGE2 and PGF2α, and appears to represent a novel signaling pathway.