The search for the causes of neurodegenerative disorders is a major theme in brain research. Acquired disturbances of several aspects of cellular metabolism appear pathologically important in sporadic Alzheimer's disease (SDAT). Among these brain glucose utilisation is reduced in the early stages of the disease and the regulatory enzymes important for glucose metabolism are reduced. In the brain, insulin, insulin-like growth factors and their receptors regulate glucose metabolism and promote neuronal growth. To detect changes in the functional activity of the brain insulin neuromodulatory system of SDAT patients, we determined the concentrations of insulin and c-peptide as well as insulin receptor binding and IGF-I receptor binding in several regions of postmortem brain cortex during aging and Alzheimer's disease. Additionally, we performed immunohistochemical staining with antibodies against insulin in neocortical brain areas in SDAT and controls. We show for the first time that insulin and c-peptide concentration in the brain are correlated and decrease with aging, as do brain insulin receptor densities. Weak insulin-immunoreactivity could be demonstrated histochemically in pyramidal neurons of controls, whereas in SDAT a stronger insulin-immunoreactivity was found. On a biochemical level, insulin and c-peptide levels were reduced compared to middle-aged controls, but were unchanged compared to age-matched controls. Brain insulin receptor densities in SDAT were decreased compared to middle-aged controls, but increased in comparison to age-matched controls. IGF-I receptor densities were unchanged in aging and in SDAT. Tyrosine kinase activity, a signal transduction mechanism common to both receptor systems, was reduced in SDAT in comparison to middle-aged and age-matched control groups. These data are consistent with a neurotrophic role of insulin in the human brain and a disturbance of insulin signal transduction in SDAT brain and favor the hypothesis that insulin dependent functions may be of pathogenetic relevance in sporadic SDAT.

Phosducin regulates Gbetagamma-stimulated signaling by binding to Gbetagamma subunits of heterotrimeric G-proteins. Control of phosducin activity by phosphorylation is well established. However, little is known about other mechanisms that may control phosducin activity. Here we report that phosducin is regulated at the posttranslational level by modification with the small ubiquitin-related modifier, SUMO. We demonstrate modification with SUMO for phosducin in vitro expressed in cells and for native phosducin purified from retina and the heart. A consensus motif for SUMOylation was identified in phosducin at amino acid positions 32-35. Mutation of the conserved lysine 33 to arginine in this motif abolished SUMOylation of phosducin, indicating that SUMO is attached to lysine 33 of phosducin. In transfected cells the steady-state levels of the K33R mutant protein were much lower compared with wild-type phosducin. The investigation of the stability of wild-type phosducin and of phosducinK33R showed a decreased protein stability of the SUMOylation-deficient mutant. The decreased protein stability correlated with increased ubiquitinylation of the SUMOylation-deficient mutant. These findings indicate that SUMOylation protects phosducin from proteasomal degradation. SUMOylation of phosducin decreased its ability to bind Gbetagamma. PhlP, a closely related member of the phosducin family, was not a target for SUMOylation, but its SUMOylation can be achieved by a single amino acid insertion in the conserved N terminus of PhlP. Together, these findings show that phosducin is a previously unrecognized target of SUMO modification and that SUMOylation controls phosducin stability in cells as well as its functional properties.

G protein-coupled receptor kinase 2 (GRK2) is able to phosphorylate a variety of agonist-occupied G proteincoupled receptors (GPCR) and plays an important role in GPCR modulation. However, recent studies suggest additional cellular functions for GRK2. Phosducin and phosducin-like protein (PhLP) are cytosolic proteins that bind G␤␥ subunits and act as regulators of G-protein signaling. In this report, we identify phosducin and PhLP as novel GRK2 substrates. The phosphorylation of purified phosducin and PhLP by recombinant GRK2 proceeds rapidly and stoichiometrically (0.82 ؎ 0.1 and 0.83 ؎ 0.09 mol of P i /mol of protein, respectively). The phosphorylation reactions exhibit apparent K m values in the range of 40 -100 nM, strongly suggesting that both proteins could be endogenous targets for GRK2 activity. Our data show that the site of phosducin phosphorylation by GRK2 is different and independent from that previously reported for the cAMP-dependent protein kinase. Analysis of GRK2 phosphorylation of a variety of deletion mutants of phosducin and PhLP indicates that the critical region for GRK2 phosphorylation is localized in the C-terminal domain of both phosducin and PhLP (between residues 204 and 245 and 195 and 218, respectively). This region is important for the interaction of these proteins with G␤␥ subunits. Phosphorylation of phosducin by GRK2 markedly reduces its G␤␥ binding ability, suggesting that GRK2 may modulate the activity of the phosducin protein family by disrupting this interaction. The identification of phosducin and PhLP as new substrates for GRK2 further expands the cellular roles of this kinase and suggests new mechanisms for modulating GPCR signal transduction.Agonist occupancy of G protein-coupled receptors (GPCR) 1 triggers interaction with different types of cellular proteins. Interaction with heterotrimeric G proteins promotes GTP/GDP exchange and dissociation into G␣ and G␤␥ subunits, both of which modulate different membrane-bound effector proteins. This signal transduction process is regulated at different levels. Agonist-activated GPCRs are phosphorylated by a family of specific G protein-coupled receptor kinases (GRKs). This is followed by binding of regulatory proteins termed arrestins to the phosphorylated receptor leading to uncoupling from G proteins, a process known as desensitization (reviewed in Refs.

Phosducin-like protein (PhLP) is a member of the phosducin family of G-protein ␤␥-regulators and exists in two splice variants. The long isoform PhLP L and the short isoform PhLP S differ by the presence or absence of an 83-amino acid N terminus. In isolated biochemical assay systems, PhLP L is the more potent G␤␥-inhibitor, whereas the functional role of PhLP S is still unclear. We now report that in intact HEK 293 cells, PhLP S inhibited G␤␥-induced inositol phosphate generation with ϳ20-fold greater potency than PhLP L . Radiolabeling of transfected HEK 293 cells with [ 32 P] revealed that PhLP L is constitutively phosphorylated, whereas PhLP S is not. Because PhLP L has several consensus sites for the constitutively active kinase casein kinase 2 (CK2) in its N terminus, we tested the phosphorylation of the recombinant proteins by either HEK cell cytosol in the presence or absence of kinase inhibitors or by purified CK2. PhLP L was a good CK2 substrate, whereas PhLP S and phosducin were not. Progressive truncation and serine/threonine to alanine mutations of the PhLP L N terminus identified a serine/threonine cluster (Ser-18/ Thr-19/Ser-20) within a small N-terminal region of PhLP L (amino acids 5-28) as the site in which PhLP L function was modified in HEK 293 cells. In native tissue, PhLP L also seems to be regulated by phosphorylation because phosphorylated and non-phosphorylated forms of PhLP L were detected in mouse brain and adrenal gland. Moreover, the alternatively spliced isoform PhLP S was also found in adrenal tissue. Therefore, the physiological control of G-protein regulation by PhLP seems to involve phosphorylation by CK2 and alternative splicing of the regulator.

S but did not lead to a functional rescue. Moreover, in the presence of PhLP S , stabilized G␥ subunits did not coprecipitate with stabilized G␤ subunits, suggesting that PhLP S might interfere with G␤␥ folding. PhLP S and several truncated mutants of PhLP S interacted with the subunit tailless complex polypeptide-1␣ (TCP-1␣) of the CCT chaperonin complex, which is involved in protein folding. Knock-down of TCP-1␣ in HEK cells by small interfering RNA also led to down-regulation of G␤␥. We therefore conclude that the strong inhibitory action of PhLP S on G␤␥ signaling is the result of a previously unrecognized mechanism of G␤␥-regulation, inhibition of G␤␥-folding by interference with TCP-1␣.The two splice variants of phosducin-like protein (PhLP)