In rod photoreceptor cells, Ca 2+ ‐bound recoverin associates with disk membranes and inhibits light‐dependent phosphorylation of rhodopsin. However, the functional significance of Ca 2+ ‐induced membrane association of recoverin has not been fully evaluated. We found that Ca 2+ ‐bound recoverin forms a complex with rhodopsin kinase preferentially at the membrane surface. Addition of increasing amounts of membranes promoted the membrane association of recoverin, and remarkably suppressed rhodopsin kinase activity. It was concluded that the Ca 2+ ‐recoverin‐rhodopsin kinase complex is stabilized by membrane association, leading to effective suppression of the kinase activity.
Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.
Muscarinic acetylcholine receptors (mAChR, human m2 subtype) expressed in Sf9 (Spodoptera frugiperda ) cells using the baculovirus system were purified and subjected to phosphorylation by a mAChR kinase, which was partially purified from porcine cerebrum. Two bands with apparent molecular masses of 59 kDa and 39 kDa as determined by SDS/PAGE were found to be phosphorylated in an agonist‐dependent manner. Both bands were labeled by the irreversible muscarinic ligand [ 3 H]propylbenzilylcholine mustard. Molecular masses of the [ 32 P]phosphorylated or [ 3 H]propylbenzilylcholine‐mustard‐labeled bands decreased following treatment with N ‐glycanase. The 59‐kDa and 39‐kDa bands were converted to 52‐kDa bands 32‐kDa bands, respectively, indicating that both the 59‐kDa and 39‐kDa bands contain the amino‐terminal region where glycosylation sites are present. The ratio of incorporated [ 32 P]phosphate and bound [ 3 H]propylbenzilylcholine mustard was essentially the same for the 59‐kDa and 39‐kDa bands, indicating that all the phosphorylation sites reside in the sequence of 39 kDa from the amino‐terminal region. The amounts of incorporated [ 32 P]phosphate were estimated to be 10–11/receptor, with 7–8 serine and 3–4 threonine, but no phosphorylated tyrosine residues. Further treatment of [ 32 P]phosphorylated or [ 3 H]propylbenzilycholine‐mustard‐labeled receptors with V8 protease indicated that the phosphorylation sites were not present in 30‐kDa amino‐terminal segment. These results indicate that the phosphorylation sites are localized in the range 30–39 kDa from the amino terminus, which consists of primarily the central part of the third intracellular loop. Consistent with this conclusion, a fusion protein containing glutathione S ‐transferase linked to a peptide corresponding to residues 227–324 of the central part of the third intracellular loop was found to be phosphorylated by the mAChR kinase in a heparin‐sensitive manner.
Abstract Addictive drugs such as amphetamine and cocaine stimulate the dopaminergic system, activate dopamine receptors and induce gene expression throughout the striatum. The signal transduction pathway leading from dopamine receptor stimulation at the synapse to gene expression in the nucleus has not been fully elucidated. Here, we present evidence that D1 receptor stimulation leads to phosphorylation of the transcription factor Ca 2+ and cyclic AMP response element binding protein (CREB) in the nucleus by means of NMDA receptor‐mediated Ca 2+ signaling. Stimulation of D1 receptors induces the phosphorylation of Ser897 on the NR1 subunit by protein kinase A (PKA). This phosphorylation event is crucial for D1 receptor‐mediated CREB phosphorylation. Dopamine cannot induce CRE‐mediated gene expression in neurons transfected with a phosphorylation‐deficient NR1 construct. Moreover, stimulation of D1 receptors or increase in cyclic AMP levels leads to an increase in cytosolic Ca 2+ in the presence of glutamate, but not in the absence of glutamate, indicating the ability of dopamine and cyclic AMP to facilitate NMDA channel activity. The recruitment of the NMDA receptor signal transduction pathway by D1 receptors may provide a general mechanism for gene regulation that is fundamental for mechanisms of drug addiction and long‐term memory.
Rab proteins play a critical role in intracellular vesicle trafficking and require post-translational modification by adding lipids at the C-terminus for proper functions. This modification is preceded by the formation of a trimeric protein complex with the Rab escort protein (REP) and the Rab geranylgeranyltransferase (RabGGTase). However, the genetic hierarchy among these proteins and the tissue-specificity of each protein function are not yet clearly understood. Here we identified the Caenorhabditis elegans rep-1 gene and found that a rep-1 mutant showed a mild defect in synaptic transmission and defecation behaviors. Genetic analyses using the exocytic Rab mutants rab-3 or rab-27 suggested that rep-1 functions only in the RAB-27 pathway, and not in the RAB-3 pathway, for synaptic transmission at neuromuscular junctions. However, the disruption of REP-1 did not cause defecation defects compared to severe defects in either RAB-27 or RabGGTase disruption, suggesting that REP-1 is not essential for RAB-27 signaling in defection. Some Rab proteins did not physically interact with REP-1, and localization of these Rab proteins was not severely affected by REP-1 disruption. These findings suggest that REP-1 functions are required in specific Rab pathways and in specific tissues, and that some Rab proteins are functionally prenylated without REP-1.
