The long term goal of this proposal is to determine the role of calmodulin (CaM) in mediating Ca2+-dependent prolactin secretion from GH3 rat anterior pituitary tumor cells. Proteinaceous CaM antagonists (anti-CaM IgG, chlorpromazine- CAM, nor2chlorpromazine-CaM and mastoparan) will be incorporated into cultured GH3 cells using erythrocyte ghosts and a new hemagglutinin fusion method. The ability of these CaM antagonists to inhibit prolactin secretion in response to 50 mM K+ or the Ca2+ ionophore, A23187, will be examined. To initiate identification of the CaM binding proteins which may modulate prolactin secretion, the MW of CaM binding proteins in GH3 cell cytosolic and particulate fractions will be determined using the photoaffinity probe, azido(125I)iodoCaM. Radiolabeling of CaM binding proteins will be compared using this technique and the (125I)CaM gel overlay procedure. The simplest method which specifically labels the most CaM binding proteins will be used to screen the ability of CaM antagonists to inhibit radiolabeling of each CaM binding protein. The CaM antagonist dose response for inhibition of radiolabeled CaM binding will be compared with dose responses for inhibition of prolactin secretion. The ability of CaM antagonists to inhibit Ca2+dependent phosphorylation of specific GH3 cell protein will be determined using 2-D gel electrophoresis and autoradiography. CaM-antagonist dose responses for inhibition of protein phosphorylation will be compared with dose responses for inhibition of radiolabeling of CaM binding proteins and with dose responses for inhibition of prolactin release. These results should allow one to determine: (1) whether CaM mediates prolactin secretion, and if so (2) which proteins it binds with to mediate this process, (3) whether specific proteins are phosphorylated by CaM-dependent process, and (4) whether CaM binding proteins and proteins phosphorylated by a CaM-dependent process appear to be associated with prolactin secretion. Because CaM is thought to mediate Ca2+-dependent secretory processes in general, these results may help to elucidate a Ca2+/CaM secretory pathway which is common to many tissues, including the pituitary, adrenal medulla, and central and peripheral nerves. Understanding such a pathway would be an integral part of treating Ca2+-dependent secretory disorders.
