| CAMP-dependent protein kinase (Protein Kinase A, PKA) is a highly conserved serine/threonine kinase that acts as a major regulator of cell biology through its modulation of enzymatic activity, ion permeability, and gene expression. The inactive heterotetrameric holoenzyme complex consists of two regulatory (R) and two catalytic (C) subunits, and activates upon dissociation induced by conformational changes in the R subunits upon binding adenosine 3 ',5'-cyclic monophosphate (cAMP). Four R subunit genes (RIalpha, RIbeta RIIalpha, RIIbeta) and two C subunit genes (Calpha and Cbeta) are present in the mouse genome, and targeted disruption of each of these genes has resulted in the analysis of PKA-mediated signaling in many physiological systems. This dissertation investigates the physiological and molecular roles of PKA in body weight regulation and male fertility.; Mice lacking the RIIbeta subunit of PKA exhibit a decrease in white adipose tissue stores (WAT), are resistant to diet-induced obesity and diabetes, and have an elevation in the brown adipose tissue-specific mitochondrial uncoupling protein 1 (Ucp1). To determine if Ucp1-dependent energy expenditure is required for the lean phenotype, RIIbeta-/- mice were placed on a Ucp1-/- background and the metabolic parameters of oxygen consumption, locomotor activity, and WAT content were examined. Ucp1 is required for the increased oxygen consumption of RIIbeta -/- mice, but is not required for the hyperactive phenotype. Surprisingly, RIIbeta/Ucp1 double knockout mice remain lean compared to control mice, demonstrating that Ucp1-dependent energy expenditure is not the sole cause of RIIbeta-/- leanness.; Mice lacking the male germ cell-specific splice variant of the Calpha gene (Calpha2) were generated to determine the signaling events downstream of HCO3--stimulated CAMP transients that require PKA in sperm. Female Calpha2-/- mice appeared completely normal, but Calpha2-/- males were infertile despite normal body size, testis weight, and sperm counts. Examination of mutant cauda epididymal sperm revealed severe motility defects, in both percent motile and flagellar waveform. HCO3- failed to enhance motility and depolarization-evoked Ca2+ entry in Calpha2-/- sperm, leading to a failure in capacitation and zona pellucida penetration. Calpha2-/- sperm also displayed elevated basal and HCO3- -stimulated cAMP generation, implicating PKA in a feedback inhibitory loop to regulate sperm adenylyl cyclase activity. |
