Role of receptor kinase in long-term desensitization of the cardiac muscarinic receptor-K+ channel system.

Desensitization of the cardiac muscarinic K+ channel was studied in cultured neonatal rat atrial cells and in Chinese hamster ovary (CHO) cells transfected with muscarinic receptor (HM(2)), G protein-coupled inward rectifying K+ channels 1 and 4, and G protein-coupled receptor kinase 2. In atrial cells incubated in 10 microM carbachol for 24 h, channel activity in cell-attached patches was substantially reduced as a result of long-term desensitization. The long-term desensitization was also observed in CHO cells transfected with the wild-type receptor and receptor kinase (as well as the channel). However, long-term desensitization was greatly reduced or abolished if the cells were 1) not transfected with the receptor kinase, 2) transfected with a mutant receptor lacking phosphorylation sites (rather than the wild-type receptor), or 3) transfected with a mutant receptor kinase lacking kinase activity (rather than the wild-type receptor kinase). We suggest that long-term desensitization of the cardiac muscarinic receptor-K+ channel system to muscarinic agonist may involve phosphorylation of the receptor by receptor kinase.

Effects of short-term toluene exposure on ligand binding to muscarinic acetylcholine receptors in the rat frontal cortex and hippocampus.

Changes in the binding affinity of the muscarinic acetylcholine receptor agonist carbamylcholine were determined in membranes isolated from the brains of rats exposed to toluene at concentrations of 500-2,000 ppm for 6 h. Membrane fractions of the frontal cortex and hippocampus were prepared and agonist-binding affinities were determined by measuring the displacement of [3H]N-methyl scopolamine-binding activity by carbamylcholine. In the frontal cortex, the affinity of high-affinity carbamylcholine binding was reduced following exposure to toluene at a concentration of 1000 ppm or higher. However, in the hippocampus, the affinity of high-affinity binding of carbamylcholine was increased following exposure to toluene. These observations suggest that toluene exposure affects binding affinity of carbamylcholine, and the effect differs by brain region.

Effects of toluene on regulation of adenylyl cyclase by stimulation of G-protein-coupled receptors expressed in CHO cells.

We determined the effects of toluene exposure on activation or inhibition of adenylyl cyclase by stimulating human beta2-adrenergic receptors (beta2-AR) and muscarinic acetylcholine receptor (mAChR) m2 subtypes, respectively, expressed in CHO cells. The formation of cAMP via beta2-AR stimulation was slightly but not significantly facilitated in the presence of 3.7 microM toluene. On the other hand, the inhibition of adenylyl cyclase by 10 microM of carbamylcholine stimulation of mAChR m2 subtypes was attenuated in the presence of toluene. These results strongly suggest that toluene affects activation of Gi rather than Gs.

Ca2+/calmodulin-mediated regulation of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells.

We investigated the regulation by intracellular Ca2+ of agonist-induced sequestration of Gq protein-coupled histamine H1 receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H1 receptors from the cell surface membrane was detected as the loss of [3H]mepyramine binding sites on intact cells accessible to the hydrophilic H1-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [3H]mepyramine were mirrored by changes in the subcellular distribution of H1 receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H1 receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca2+ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca2+/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H1-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of Gq protein-coupled receptors is transiently inhibited by Ca2+/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.

Desensitization of human muscarinic acetylcholine receptor m2 subtypes is caused by their sequestration/internalization.

Desensitization of human muscarinic acetylcholine receptor m2 subtypes (hm2 receptors) stably expressed in chinese hamster ovary cells was measured as decreases in the carbamylcholine-stimulated [35S]GTPgammaS binding activity in membrane preparations after pre-treatment of cells with carbamylcholine. The extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity was found to decrease to 64% following pretreatment of cells with 10 microM carbamylcholine for 30 min, and under the same conditions 51-59% of hm2 receptors were sequestered/internalized as assessed by decreases in the [3H]N-methylscopolamine binding activity on the cell surface. A similar reduction in the carbamylcholine-stimulated [35S]GTPgammaS binding activity was observed by pretreatment of cells with 5 nM propylbenzylylcholine mustard, which irreversibly bound to and inactivated 58% of the hm2 receptors. When the cells were pretreated with 10 microM carbamylcholine in the presence of 0.32 M sucrose, which is known to inhibit clathrin-mediated endocytosis, no sequestration/internalization of hm2 receptors was observed, and the extent of carbamylcholine-stimulated [35S]GTPgammaS binding activity did not change. These results indicate that desensitization of hm2 receptors may be caused by reduction of receptor number on the cell surface through sequestration/internalization rather than by loss of the function of receptors.

Internalization and down-regulation of human muscarinic acetylcholine receptor m2 subtypes. Role of third intracellular m2 loop and G protein-coupled receptor kinase 2.

Internalization and down-regulation of human muscarinic acetylcholine m2 receptors (hm2 receptors) and a hm2 receptor mutant lacking a central part of the third intracellular loop (I3-del m2 receptor) were examined in Chinese hamster ovary (CHO-K1) cells stably expressing these receptors and G protein-coupled receptor kinase 2 (GRK2). Agonist-induced internalization of up to 80-90% of hm2 receptors was demonstrated by measuring loss of [3H]N-methylscopolamine binding sites from the cell surface, and transfer of [3H]quinuclidinyl benzilate binding sites from the plasma membrane into the light-vesicle fractions separated by sucrose density gradient centrifugation. Additionally, translocation of hm2 receptors with endocytic vesicles were visualized by immunofluorescence confocal microscopy. Agonist-induced down-regulation of up to 60-70% of hm2 receptors was demonstrated by determining the loss of [3H]quinuclidinyl benzilate binding sites in the cells. The half-time (t1/2) of internalization and down-regulation in the presence of 10(-4) M carbamylcholine was estimated to be 9.5 min and 2.3 h, respectively. The rates of both internalization and down-regulation of hm2 receptors in the presence of 10(-6) M or lower concentrations of carbamylcholine were markedly increased by coexpression of GRK2. Agonist-induced internalization of I3-del m2 receptors was barely detectable upon incubation of cells for 1 h, but agonist-induced down-regulation of up to 40-50% of I3-del m2 receptors occurred upon incubation with 10(-4) M carbamylcholine for 16 h. However, the rate of down-regulation was lower compared with wild type receptors (t1/2 = 9.9 versus 2.3 h). These results indicate that rapid internalization of hm2 receptors is facilitated by their phosphorylation with GRK2 and does not occur in the absence of the third intracellular loop, but down-regulation of hm2 receptors may occur through both GRK2-facilitating pathway and third intracellular loop-independent pathways.

Sequestration of human muscarinic acetylcholine receptor hm1-hm5 subtypes: effect of G protein-coupled receptor kinases GRK2, GRK4, GRK5 and GRK6.

Sequestration of porcine muscarinic acetylcholine receptor m2 subtypes (m2 receptors) expressed in COS-7 cells is facilitated by coexpression of G protein-coupled receptor kinases 2 (GRK2). We examined the effect of coexpression of GRK2, GRK4 delta, GRK5 and GRK6 on sequestration of human m1-m5 receptors expressed in COS-7 cells, which was assessed as loss of [3H]N-methylscopolamine binding activity from the cell surface. Sequestration of m4 receptors as well as m2 receptors was facilitated by coexpression of GRK2 and attenuated by coexpression of the dominant negative form of GRK2 (DN-GRK2). Sequestration of m3 and m5 receptors also was facilitated by coexpression of GRK2 but not affected by coexpression of DN-GRK2. On the other hand, proportions of sequestered m1 receptors were not significantly different with coexpression of GRK2 and DN-GRK2. GRK4 delta, GRK5 and GRK6 did not facilitate sequestration of m1-m5 receptors in COS-7 cells, except that the sequestration of m2 receptors tended to be facilitated by coexpression of GRK4 delta, GRK5 and GRK6. However, coexpression of GRK4 delta, GRK5, but not GRK6, in BHK-21 cells facilitated sequestration of m2, but not m3, receptors. These results indicate that the effect of GRK2 to facilitate receptor sequestration is not restricted to m2 receptors but is generalized to other muscarinic receptors except m1 receptors and that other kinases, including GRK4 delta, GRK5 and endogenous kinase(s) in COS-7 cells, also contribute to sequestration of m2 and m4 receptors.

Role of receptor kinase in short-term desensitization of cardiac muscarinic K+ channels expressed in Chinese hamster ovary cells.

1. The cardiac muscarinic receptor-K+ channel system was reconstructed in Chinese hamster ovary (CHO) cells by transfecting the cells with the various components of the system. The activity of the muscarinic K+ channel was measured with the cell-attached configuration of the patch clamp technique. 2. In CHO cells transfected with the channel (Kir3.1/Kir3.4), receptor (hm2) and receptor kinase (GRK2), on exposure to agonist, there was a decline in channel activity as a result of desensitization, similar to that in atrial cells. 3. Whereas the desensitization was almost abolished by not transfecting with the receptor kinase or by transfecting with a mutant receptor lacking phosphorylation sites, it was only reduced (by approximately 39%) by transfecting with a mutant receptor kinase with little/kinase activity. 4. These results suggest that the receptor kinase is responsible for desensitization of the muscarinic K+ channel and that this involves phosphorylation-dependent and -independent mechanisms.

Ca2+-dependent inhibition of G protein-coupled receptor kinase 2 by calmodulin.

Agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptor m2 subtypes (m2 receptors) or rhodopsin by G protein-coupled receptor kinase 2 (GRK2) was found to be inhibited by calmodulin in a Ca2+-dependent manner. The phosphorylation was fully inhibited in the absence of G protein betagamma subunits and partially inhibited in the presence of betagamma subunits. The dose-response curve for stimulation by betagamma subunits of the m2 and rhodopsin phosphorylation was shifted to the higher concentration of betagamma subunits by addition of Ca2+-calmodulin. The phosphorylation by GRK2 of a glutathione S-transferase fusion protein containing a peptide corresponding to the central part of the third intracellular loop of m2 receptors (I3-GST) was not affected by Ca2+-calmodulin in the presence or absence of betagamma subunits, but the agonist-dependent stimulation of I3-GST phosphorylation by an I3-deleted m2 receptor mutant in the presence of betagamma subunits was suppressed by Ca2+-calmodulin. These results indicate that Ca2+-calmodulin does not directly interact with the catalytic site of GRK2 but inhibits the kinase activity of GRK2 by interfering with the activation of GRK2 by agonist-bound m2 receptors and G protein betagamma subunits. In agreement with the assumption that GRK2 activity is suppressed by the increase in intracellular Ca2+, the sequestration of m2 receptors expressed in Chinese hamster ovary cells was found to be attenuated by the treatment with a Ca2+ ionophore, A23187.

Sequestration of the short and long isoforms of dopamine D2 receptors expressed in Chinese hamster ovary cells.

The short (D2S) and long (D2L) isoforms of dopamine D2 receptors were stably expressed in Chinese hamster ovary cells, and dopamine-induced sequestration was examined by measuring the loss of binding of the hydrophilic ligand [3H]sulpiride from the cell surface. Dopamine treatment of Chinese hamster ovary cells expressing D2S for 30 min at 37 degrees caused a 43.8 +/- 3.4% decrease in [3H]sulpiride binding activity measured by incubation of the treated cells with [3H]sulpiride at 4 degrees for 4 hr after the dopamine was washed out. The half-life of the decrease in binding was estimated to be 18.7 +/- 1.6 min, and the concentration of dopamine giving a half-maximal effect (EC50) was estimated to be 180 +/- 90 nM. The decrease was reversible, and the binding activity was recovered by washing out the dopamine and incubating the cells at 37 degrees for 30 min but was not reversible when the cells were incubated at 4 degrees. The binding activity of [3H]spiperone, a hydrophobic ligand, was not affected by the dopamine treatment under the same experimental conditions. These results indicate that approximately one half of the D2S receptors undergo agonist-induced sequestration, probably endocytosis, in a reversible and temperature-dependent manner. Sequestration of D2L receptors was not as apparent as that of D2S receptors; the decrease in [3H]sulpiride binding activity was 21.6 +/- 0.9% and the rate of the decrease was delayed, with a half-life of 33.2 +/- 7.8 min, although effective concentrations of dopamine were similar, with EC50 = 170 +/- 50 nM. A D2S receptor variant containing a missense mutation changing Ser311 in the third intracellular loop to cysteine was found to be sequestered to a significantly lesser extent than with wild-type D2S receptors. This finding was discussed with respect to the report that this variant gene is found more frequently in schizophrenic patients than in control subjects.

Carbachol-induced desensitization of rat basophilic leukemia (RBL-2H3) cells transfected with human m3 muscarinic acetylcholine receptors.

1. Carbachol-induced homologous desensitization of the secretory response was investigated by transfecting RBL-2H3 cells with cDNA encoding the human m3 muscarinic acetylcholine receptor (RBL-m3). 2. Exposure of RBL-m3 cells to 100 microM carbachol for 30 min in Ca2+-free medium inhibited the secretion induced by the subsequent addition of 10 microM carbachol plus Ca2+. 3. Desensitized cells bound [3H]quinuclidinyl benzilate with a similar Bmax and Kd to those of control cells. 4. The carbachol-induced transient increase in levels of inositol 1,4,5-trisphosphate was not changed by desensitization. 5. Homologous desensitization persisted when desensitized cells were permeabilized with Staphylococcal alpha-toxin.

Sequestration of muscarinic acetylcholine receptor m2 subtypes. Facilitation by G protein-coupled receptor kinase (GRK2) and attenuation by a dominant-negative mutant of GRK2.

Sequestration of m2 receptors (muscarinic acetylcholine receptor m2 subtypes), which was assessed as loss of N-[3H]methylscopolamine ([3H]NMS) binding activity from the cell surface, was examined in COS 7 and BHK-21 cells that had been transfected with expression vectors encoding the m2 receptor and, independently, vectors encoding a G protein-coupled receptor kinase (GRK2) (beta-adrenergic receptor kinase 1) or a GRK2 dominant-negative mutant (DN-GRK2). The sequestration of m2 receptors became apparent when the cells were treated with 10(-5) M or higher concentrations of carbamylcholine. In this case, approximately 40% or 20-25% of the [3H]NMS binding sites on COS 7 or BHK-21 cells, respectively, were sequestered with a half-life of 15-25 min. In cells in which GRK2 was also expressed, the sequestration became apparent in the presence of 10(-7) M carbamylcholine. Approximately 40% of the [3H]NMS binding sites on both COS 7 and BHK-21 cells were sequestered in the presence of 10(-6) M or higher concentrations of carbamylcholine. When DN-GRK2 was expressed in COS 7 cells, the proportion of [3H]NMS binding sites sequestered in the presence of 10(-5) M or higher concentrations of carbamylcholine was reduced to 20-30%. These results indicate that the phosphorylation of m2 receptors by GRK2 facilitates their sequestration. These results are in contrast with the absence of a correlation between sequestration and the phosphorylation of beta-adrenergic receptors by the GRK2 and suggests that the consequences of phosphorylation by GRK2 are different for different receptors.