ABSTRACT
In past reports we illustrated the importance of Y131, Y322, and T137 within the intracellular (IC) face of the rat bradykinin B2 receptor (rBKB2R) for signal transduction and receptor maintenance (Prado et al. [1997] J. Biol. Chem. 272:14638-14642; Prado et al. [1998] J. Biol. Chem. 273:33548-33555). In this report, we mutate the remaining hydroxyl possessing residues located within the rBKB2R IC region. Exchange of S139A (IC2) or T239V (IC3) did not affect BK activated phosphatidylinositol (PI) turnover or receptor internalization. Chimeric exchange of the last 34 amino acids of BKB2R C-terminus with the corresponding 34 amino acids of the rat angiotensin II AT1a receptor (rAT1aR), both containing an S/T cluster, resulted in a mutant with normal endocytosis and BK activated PI turnover. A more selective chimera of these S/T clusters, with an exchange of BKB2R (333-351) with a rAT1aR fragment (326-342), resulted in a receptor with a retarded internalization but a normal BK activated PI turnover. Subsequent mutation of rBKB2R T344V showed little change in receptor uptake but a pronounced loss of BK activated PI turnover. The mutation of S335A, S341A, S348A, and S350A resulted in very poor receptor internalization and loss of activated PI turnover. Closer examination of this serine cluster illustrated that the replacement of S348A led to poor internalization; whereas the retention of S348 and mutation of S341A resulted in a receptor with a much greater internalization than WT. These and other results suggest that the presence of S348 promotes internalization while the presence of S341 dampens it. Conversely, S341 and S350 proved important for receptor signaling. In sum, our results illustrate that the distal C-terminus including its S/T cluster is important for both rBKB2R internalization and signal transduction. Individual S/T residues within this cluster appear involved in either signal transmission or receptor uptake capacity. However, replacement of the entire distal tail region with the corresponding rAT1aR sequence, also containing an S/T cluster, enables the BKB2R/AT1aR chimera to act in a very similar manner to wild type rBKB2R.
Subject(s)
Hydroxyl Radical , Receptors, Angiotensin/chemistry , Receptors, Angiotensin/genetics , Receptors, Bradykinin/chemistry , Receptors, Bradykinin/genetics , Signal Transduction , Amino Acid Sequence , Amino Acids/chemistry , Animals , Cell Line , DNA Primers/pharmacology , Endocytosis , Microscopy, Fluorescence , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Phosphatidylinositols/metabolism , Protein Structure, Tertiary , Rats , Receptor, Bradykinin B2 , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Time Factors , TransfectionABSTRACT
Purkinje cell degeneration (pcd) mutant mice lose virtually all Purkinje cells. It was found that the pcd mutants did not show within-sessions habituation in terms of hole-poking responses. This type of perseverative responding has been found in rats with limbic system damage and implies a role for the cerebellum in behavioral inhibition.
Subject(s)
Cerebellum/physiology , Exploratory Behavior/physiology , Habituation, Psychophysiologic/physiology , Mice, Neurologic Mutants/physiology , Purkinje Cells/physiology , Animals , Female , Male , MiceABSTRACT
Weaver mutant mice engaged less in motor activity and hole poking. Weaver mice alternated spontaneously in a 4-trial but not a 2-trial test. These results are similar to those of a previous study with staggerer mutants, in that both are less active. However, weaver mutants are less affected in the spontaneous alternation measure.
Subject(s)
Cerebellum/physiology , Corpus Striatum/physiology , Discrimination Learning/physiology , Exploratory Behavior/physiology , Orientation/physiology , Receptors, Dopamine/physiology , Animals , Mice , Mice, Neurologic Mutants , Neurons/physiologyABSTRACT
Staggerer mutant mice were found to be less active in terms of motor activity in a maze, hole poking and rearing. Staggerer mutant mice also showed a lack of spontaneous alternation in both 2-trial and 4-trial tests. A lack of spontaneous alternation in this mutant may be due to a deficit in response inhibition or in spatial orientation, similar to that of animals with limbic lesions.
Subject(s)
Cerebellum/physiology , Mice, Neurologic Mutants/physiology , Motor Activity/physiology , Animals , Inhibition, Psychological/physiology , Mice , Space Perception/physiologyABSTRACT
Purkinje cell degeneration (pcd) mutant mice lose cerebellar Purkinje cells. It was found that pcd mice, contrary to normal mice, did not alternate spontaneously at any of 3 inter-trial intervals (15 s, 3 or 6 min). Deficits in spontaneous alternation are typical of animals with brain damage in areas important in spatial learning. Results are discussed in terms of a role for the cerebellum in spatially mediated behavior and behavioral inhibition.
Subject(s)
Mice, Neurologic Mutants/physiology , Nerve Degeneration , Purkinje Cells/physiology , Spatial Behavior/physiology , Animals , Cerebellum/physiology , Mice , Mice, Inbred C57BLABSTRACT
Purkinje cell degeneration (pcd) mutant mice were found to be as active as normal mice in a T-maze. The pcd mutants, contrary to normal mice, did not alternate spontaneously in either a 2-trial or a 4-trial test. Results are discussed in terms of a role for the cerebellum in behavioral inhibition and visuo-spatial organization.
