Specific Identification and Subcellular Localization of Three Calmodulin-binding Proteins in the Rat Gonadotrope: Spectrin, Caldesmon, and Calcineurin1

In an effort to characterize the second messenger system for LH release, we have previously identified five calmodulin-binding proteins in rat gonadotropes of Mr greater than 205,000, 200,000, 135,000, 60,000, and 52,000. In the present study, we have used a calmodulin overlayer assay combined with Western blotting to determine the molecular identity of three calmodulin-binding proteins in rat gonadotropes: the alpha subunit of spectrin (Mr greater than 205,000), caldesmon (Mr 84,000), and the alpha subunit of calcineurin (Mr 60,000). The Mr greater than 205,000 and Mr 60,000 components or rat pituitary which bind calmodulin are immunoreactive with spectrin and calcineurin antisera, respectively. Rat pituitary also contains an Mr 84,000 component, which is immunoreactive with polyclonal sera and monoclonal antibody raised to chicken gizzard caldesmon (Mr 150,000). Like caldesmon from other sources, the Mr 84,000 component remains soluble after heat treatment and preferentially binds either filamentous actin or calmodulin, depending on the Ca2+ concentration. The three calmodulin-binding proteins were localized specifically in gonadotropes using indirect immunofluorescence microscopy or by Western-blotting cell fractions enriched for gonadotropes. After differential centrifugation of pituitary homogenate, spectrin immunoreactivity was found associated with the nuclear and secretory granule fractions, whereas caldesmon immunoreactivity was seen in the cytosolic fraction and calcineurin in the cytosolic and nuclear fractions. Although the precise role for these proteins remains unknown, the apparent requirement for calmodulin and the small number of calmodulin-binding proteins in the gonadotrope suggest their involvement in mediating GnRH actions.