Transgenic mice expressing human amyloid precursor proteins (hAPP) and amyloid-␤ peptides (A␤) in neurons develop phenotypic alterations resembling Alzheimer's disease (AD). The mechanisms underlying cognitive deficits in AD and hAPP mice are largely unknown. We have identified two molecular alterations that accurately reflect AD-related cognitive impairments. Learning deficits in mice expressing familial AD-mutant hAPP correlated strongly with decreased levels of the calcium-binding protein calbindin-D 28k (CB) and the calcium-dependent immediate early gene product c-Fos in granule cells of the dentate gyrus, a brain region critically involved in learning and memory. These molecular alterations were age-dependent and correlated with the relative abundance of A␤1-42 but not with the amount of A␤ deposited in amyloid plaques. CB reductions in the dentate gyrus primarily reflected a decrease in neuronal CB levels rather than a loss of CB-producing neurons. CB levels were also markedly reduced in granule cells of humans with AD, even though these neurons are relatively resistant to AD-related cell death. Thus, neuronal populations resisting cell death in AD and hAPP mice can still be drastically altered at the molecular level. The tight link between A␤-induced cognitive deficits and neuronal depletion of CB and c-Fos suggests an involvement of calcium-dependent pathways in AD-related cognitive decline and could facilitate the preclinical evaluation of novel AD treatments.

A-Kinase Anchoring Proteins (AKAPs) orchestrate and synchronize cellular events by tethering the cAMP-dependent protein kinase (PKA) and other signaling enzymes to organelles and membranes. The control of kinases and phosphatases that are held in proximity to activators, effectors, and substrates favors the rapid dissemination of information from one cellular location to the next. This article charts the inception of the PKA-anchoring hypothesis, the characterization of AKAPs and their nomenclature, and the physiological roles of context-specific AKAP signaling complexes.

The most frequent human apolipoprotein (apo) E isoforms, E3 and E4, differentially affect Alzheimer's disease (AD) risk (E4 > E3) and age of onset (E4 < E3). Compared with apoE3, apoE4 promotes the cerebral deposition of amyloid beta (Abeta) peptides, which are derived from the amyloid precursor protein (APP) and play a central role in AD. However, it is uncertain whether Abeta deposition into plaques is the main mechanism by which apoE isoforms affect AD. We analyzed murine apoE-deficient transgenic mice expressing in their brains human APP (hAPP) and Abeta together with apoE3 or apoE4. Because cognitive decline in AD correlates better with decreases in synaptophysin-immunoreactive presynaptic terminals, choline acetyltransferase (ChAT) activity, and ChAT-positive fibers than with plaque load, we compared these parameters in hAPP/apoE3 and hAPP/apoE4 mice and singly transgenic controls at 6-7, 12-15, and 19-24 months of age. Brain aging in the context of high levels of nondeposited human Abeta resulted in progressive synaptic/cholinergic deficits. ApoE3 delayed the synaptic deficits until old age, whereas apoE4 was not protective at any of the ages analyzed. Old hAPP/apoE4 mice had more plaques than old hAPP/apoE3 mice, but synaptic/cholinergic deficits preceded plaque formation in hAPP/apoE4 mice. Moreover, despite their different plaque loads, old hAPP/apoE4 and hAPP/apoE3 mice had comparable synaptic/cholinergic deficits, and these deficits were found not only in the hippocampus but also in the neocortex, which in most mice contained no plaques. Thus, apoE3, but not apoE4, delays age- and Abeta-dependent synaptic deficits through a plaque-independent mechanism. This difference could contribute to the differential effects of apoE isoforms on the risk and onset of AD.

To study phosphorylation of the endogenous type I thyrotropin-releasing hormone receptor in the anterior pituitary, we generated phosphosite-specific polyclonal antibodies. The major phosphorylation site of receptor endogenously expressed in pituitary GH3 cells was Thr 365 in the receptor tail; distal sites were more phosphorylated in some heterologous models. ␤-Arrestin 2 reduced thyrotropin-releasing hormone (TRH)-stimulated inositol phosphate production and accelerated internalization of the wild type receptor but not receptor mutants where the critical phosphosites were mutated to Ala. Phosphorylation peaked within seconds and was maximal at 100 nM TRH. Based on dominant negative kinase and small interfering RNA approaches, phosphorylation was mediated primarily by G protein-coupled receptor kinase 2. Phosphorylated receptor, visualized by immunofluorescence microscopy, was initially at the plasma membrane, and over 5-30 min it moved to intracellular vesicles in GH3 cells. Dephosphorylation was rapid (t1 ⁄2 ϳ 1 min) if agonist was removed while receptor was at the surface. Dephosphorylation was slower (t1 ⁄2 ϳ 4 min) if agonist was withdrawn after receptor had internalized. After agonist removal and dephosphorylation, a second pulse of agonist caused extensive rephosphorylation, particularly if most receptor was still on the plasma membrane. Phosphorylated receptor staining was visible in prolactin-and thyrotropin-producing cells in rat pituitary tissue from untreated rats and much stronger in tissue from animals injected with TRH. Our results show that the TRH receptor can rapidly cycle between a phosphorylated and nonphosphorylated state in response to changing agonist concentrations and that phosphorylation can be used as an indicator of receptor activity in vivo.The type I thyrotropin-releasing hormone (TRH) 2 receptor is a seven-transmembrane G protein-coupled receptor (GPCR) that is expressed in the anterior pituitary, where it responds to TRH secreted from the hypothalamus, leading to the release of thyroid-stimulating hormone (TSH) from thyrotrophs, which in turn causes release of T 3 and T 4 from the thyroid. Thyroid hormones exert negative feedback at the level of the hypothalamus and pituitary in order to ensure that hormone levels are tightly regulated. Lactotrophs in the anterior pituitary also express TRH receptors and release prolactin after exposure to TRH. In addition to the anterior pituitary, the TRH receptor is expressed in various areas of the central nervous system. The TRH receptor signals via G␣ q/11 , which activates phospholipase C␤ and leads to the generation of inositol 1,4,5-trisphosphate and diacylglycerol, the release of calcium from the endoplasmic reticulum, and the activation of protein kinase C (PKC).GPCRs can be phosphorylated by second messenger-activated kinases, such as PKC, or by GPCR kinases (GRKs), which preferentially recognize the agonist-occupied receptor conformation (1, 2). Like other GPCRs, the TRH receptor is phosphorylated after binding to agonist (3, 4), whi...

Protein kinase A (PKA) is activated during sympathetic stimulation of the heart and phosphorylates key proteins involved in cardiac Ca 2+ handling, including the L-type Ca 2+ channel (Ca V 1.2) and phospholamban (PLN). This results in acceleration and amplification of the beat-to-beat changes in cytosolic Ca 2+ in cardiomyocytes and, in turn, an increased rate and force of contraction. PKA is held in proximity to its substrates by protein scaffolds called A kinase anchoring proteins (AKAPs). It has been suggested that the short and long isoforms of AKAP7 (also called AKAP15/18) localize PKA in complexes with Ca V 1.2 and PLN, respectively. We generated an AKAP7 KO mouse in which all isoforms were deleted and tested whether Ca 2+ current, intracellular Ca 2+ concentration, or Ca 2+ reuptake were impaired in isolated adult ventricular cardiomyocytes following stimulation with the β-adrenergic agonist isoproterenol. KO T he key determinants of cardiac output-the force of contraction and rate of relaxation-are rooted in the amplitude and kinetics of Ca 2+ transients that occur in individual cardiomyocytes. Adrenergic stimulation initiates cAMP-dependent signaling pathways that activate PKA leading to phosphorylation of numerous proteins that are critical for Ca 2+ entry, release, and reuptake, as well as sarcomeric proteins more closely associated with contraction, such as myosin-binding protein C and troponin I. This phosphorylation amplifies Ca 2+ influx through voltagegated Ca 2+ channels (Ca V 1.2 in the ventricle) and the corresponding increase in Ca 2+ -induced Ca 2+ release from the sarcoplasmic reticulum (SR) through ryanodine receptors augments contractility. Equally important is the enhanced removal of Ca 2+ from the cytosol that allows the heart to relax more quickly during diastole, which is accomplished primarily by phosphorylating phospholamban (PLN), which in turn relieves PLN inhibition of the sarcoplasmic reticulum Ca 2+ ATPase (SERCA). Distinct, localized actions of PKA are coordinated in two ways: (i) cAMP production and hydrolysis are restricted by the subcellular localization of cyclases and phosphodiesterases, respectively, and (ii) PKA is directed to specific subcellular sites by binding to an assortment of protein scaffolds known as A kinase anchoring proteins (AKAPs) (1). By directing PKA to specific subcellular sites, AKAPs determine not only the specificity of protein phosphorylation, but also the speed with which these systems respond to adrenergic stimulation. Some AKAPs are implicated in clinically relevant cardiac signaling events (2-4). For example, regulation of potassium channel current in the heart depends on formation of complexes containing AKAP9 (yotiao), PKA, and the I Ks potassium channel α subunit (KCNQ1); an inherited single point mutation in AKAP9 impairs AKAP9-KCNQ1 interaction and ultimately leads to long QT syndrome (4).AKAP7 is expressed as a family of alternatively spliced anchoring proteins that bind all isoforms of PKA regulatory subunit, albeit with different aff...