Heart disease is one of the major cause of death in diabetic patients, but the pathogenesis of diabetic cardio-myopathy remains unclear. In this experiment, to assess the significance of G protein signaling pathways in the pathogenesis of diabetic cardiomyopathy, we analyzed the expression of G proteins and the activities of second messenger dependent protein kinases: cAMP-dependent protein kinase (PKA), DAG-mediated protein kinase C (PKC), and calmodulin dependent protein kinase II (CaM kinase II) in the streptozotocin induced diabetic rat heart. The expression of Gα α α αq was increased by slightly over 10% (P<0.05) in diabetic rat heart, while Gα α α αs, Gα α α αi, and Gβ β β β remained unchanged. The PKA activity in the heart did not change significantly but increased by 27% (P<0.01) in the liver. Insulin treatment did not restore the increased activity in the liver. Total PKC activity in the heart was increased by 56% (P<0.01), and insulin treatment did not restore such increase. The CaM kinase II activity in the heart remained at the same level but was slightly increased in the liver (14% increase, P<0.05). These findings of increased expression of Gα α α αq in the streptozotocin-diabetic rat heart that are reflected by the increased level of PKC activity and insensitivity to insulin demonstrate that alteration of Gα α α αq may underlie, at least partly, the cardiac dysfunction that is associated with diabetes.
IntroductionHeterotrimeric guanine nucleotide binding regulatory proteins (G proteins) transduce extracellular signals such as hormones, neurotransmitters and photons into intracellular signals by receptor-effector coupling. When the G protein binds to the ligand-activated receptor, it replaces the bound GDP with GTP. The GTP-bound activated G protein regulates effectors such as enzymes and ion channels (Gilman, 1987;Kaziro et al., 1991; Neer, 1995). Because most of the receptors coupled to G proteins are integral proteins that have seven hydro-phobic transmembrane domains, the G proteins need to have a membrane binding capacity to receive signals from the membrane receptors. The α subunit of G protein is known to bind tightly to the cytoplasmic face of the plasma membrane without any membrane spanning hydrophobic domain. The β γ subunit complex has a high affinity for the membrane because of isoprenylation of the gamma subunit, and plays an important role in anchoring the α subunit of G proteins (Sternweis, 1986). However, fatty acylation with myristic acid and palmitoylation was found to be important for membrane binding by α subunits of the G protein family (Jones et al., 1990, Mumby, 1997. The reversible and dynamic palmitoylation had been claimed to be critical in membrane binding by the alpha s u b u n i t of the stimulatory G protein (Gsα) (Wedegaertner et al., 1993;Wedegaertner and Bourne, 1994), but it was also argued that this domain of Gsα was not essential for membrane binding from the finding that Gsα m u t a n t s deleted in this domain retained membrane binding capacity (Degtyarev et al. , 1993;Juhnn, 1993). Thus it is necessary to make clear the importance of this modification in the binding of Gsα.The present study was performed to evaluate the role of palmitoylation in membrane binding of Gsα. We constructed Gsα mutants by replacing the glycine-2, the cognate site of myristoylation in Giα with alanine, and cysteine-3, the site of palmitoylation with serine, and then analyzed the intracellular distribution of the mutant proteins expressed in COS-1 cells by quantitative EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 30, No 4, 235-239, December 1998 Jung Abbreviations: G protein, guanine nucleotide-binding regulatory protein; Gi, adenylyl cyclase inhibitory G protein; Gs, adenylyl cyclase stimulatory G protein; Gsα, α subunit of Gs; Gα,α subunit of G protein ; Gβγ, βγ subunit of G protein Retainment of membrane binding capacity of nonpalmitoylated Gs mutants expressed in COS-1 cells
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