Protein Kinase A Anchoring to the Myometrial Plasma Membrane Is Required for Cyclic Adenosine 3',5'-Monophosphate Regulation of Phosphatidylinositide Turnover

Abstract

The importance of the localization of protein kinase A (PKA) to the plasma membrane for cAMP-mediated inhibition of phosphatidylinositide turnover was tested in an immortalized pregnant human myometrial (PHM1-41) cell line, and the putative A kinase anchoring protein (AKAP) involved was identified. Preincubation in PHM1-41 cells with chlorophenylthio-cAMP (CPT-cAMP), forskolin, or relaxin inhibited the ability of oxytocin to stimulate phosphatidylinositide turnover. The addition of a peptide that specifically disrupts interactions of PKA RII subunits with AKAPs (S-Ht31) reversed the effects of these agents, whereas a control peptide was ineffective. The pharmacology of S-Ht31 on this particular membrane event was further characterized. A 10-min incubation with S-Ht31 at a concentration of 1 microM completely reversed the inhibitory effect of relaxin on phosphatidylinositide turnover. S-Ht31 inhibited cAMP-stimulated PKA activity in PHM1-41 cell plasma membranes and decreased the concentration of PKA. Overlay analysis detected a single AKAP of approximately 86 kDa associated with the plasma membrane of PHM1-41 cells, suggesting that the association of PKA with this AKAP is important for the cAMP inhibitory mechanism. The mol wt of this AKAP was similar to that of an AKAP associated with the plasma membrane in the human brain, AKAP79. Antibodies against AKAP79 recognized a band at 86 kDa in purified plasma membranes from the PHM1-41 cells, indicating similar determinants in these proteins. These data suggest that PKA is anchored to the myometrial plasma membrane through association with an AKAP similar to AKAP79, and that this anchoring is required for the cAMP-mediated inhibition of phosphatidylinositide turnover in PHM1-41 cells.

Development of Platelet Inhibition by cAMP during Megakaryocytopoiesis

“…Also, the steps in signaling cascades initiated by surface receptors may be subject to PKA inhibition via anchorage to AKAPs. In myometrial cells, the anchoring of PKA to the plasma membrane via AKAP79 was necessary to inhibit phosphatidylinositide turnover (38). PKA is targeted to phospholipase C␤ via its regulatory subunit II␤ and AKAP79 in myometrial cells (38) and to receptors in the plasma membrane via AKAP250/gravin in endothelial cells (39,40).…”

Hormonal signaling and signal pathway crosstalk in the control of myometrial calcium dynamics

“…OTXR can exist in and outside of membrane microdomains, and signaling to different pathways is determined by location [97,98]. The inhibitory effect of PKA on PI turnover in myometrium is AKAPdependent [99]. The PKA/plasma membrane AKAP79 association decreases markedly near term in rat myometrium, coincident with a decrease in inhibitory effects of cAMP on contractant-stimulated PI turnover that occurs in the membrane [100].…”

International Union of Pharmacology LVII: Recommendations for the Nomenclature of Receptors for Relaxin Family Peptides

“…Increased intracellular cAMP was observed in mouse pubic symphysis (Braddon, 1978), in rat uterus (Cheah and Sherwood, 1980;Judson et al, 1980;Sanborn et al, 1980), and in cultures of rat (Hsu et al, 1985) and rhesus monkey myometrium (Kramer et al, 1990), human endometrium (Chen et al, 1988;Fei et al, 1990), rat anterior pituitary cell (Cronin et al, 1987), and human THP-1 monocytes (Parsell et al, 1996). The increases in cAMP are important components in relaxininduced myometrial inhibition in the rat (Dodge et al, 1999) and decidualization of human endometrial stromal cells (Huang et al, 1987;Tabanelli et al, 1992).…”