The mechanism by which muscarinic receptors internalize upon agonist exposure is poorly understood. To determine the endocytic pathways responsible for muscarinic receptor internalization, we have stably transfected human embryonic kidney (HEK 293) cells with the Hm1 (human muscarinic subtype 1) receptor tagged at the amino terminus with the epitope EYMPME. The subcellular location of the receptor was visualized by immunofluorescence confocal microscopy and quantified with the use of binding studies. The receptor redistributed into intracellular compartments following agonist treatment. This process was reversible upon removal of agonist and inhibited by antagonist. Acid treatment of the cells, which disrupts internalization via clathrin-coated vesicles, inhibited carbachol-stimulated internalization. Phorbol 12-myristate 13-acetate, on the other hand, which inhibits caveolae-mediated endocytosis, had no effect on carbachol-induced endocytosis. Double-labeling confocal microscopy was used to characterize the intracellular vesicles containing Hm1 receptor following agonist treatment. The Hm1 receptor was shown to be colocalized with clathrin and ␣-adaptin, a subunit of the AP2 adaptor protein which links endocytosed proteins with clathrin in the intracellular vesicles. In addition, endosomes containing Hm1 also contained the transferrin receptor, which internalizes via clathrin-coated vesicles. In contrast, caveolin, the protein that comprises caveolae, did not colocalize with Hm1 in intracellular vesicles following agonist treatment, indicating that caveolae are not involved in the agonist-induced internalization of Hm1. These results indicate that agonist-induced internalization of the Hm1 receptor occurs via clathrin-coated vesicles in HEK cells.Upon agonist treatment, many cell surface receptors undergo endocytosis into compartments inaccessible to extracellular ligands. This process is known as receptor internalization. The mechanism of internalization and its role in receptor regulation and function are largely unknown. Internalization may be a possible mechanism of receptor desensitization (i.e. reduction in agonist-induced activity), or alternatively, as recently suggested by Pippig et al.(1), resensitization. The endocytosed receptors may be either recycled back to the cell surface (2) or transported to lysosomes where receptors are subsequently degraded (2), a process known as receptor downregulation.Cell surface proteins may internalize into clathrin-coated vesicles (3), caveolae (4), or noncoated vesicles (5, 6). Little is known about the mechanism of internalization for the family of G protein-coupled receptors. Studies thus far suggest that the mechanism may vary with the receptor type. Raposo et al. (7) showed that noncoated vesicles appear to be involved in the endocytosis of muscarinic receptors in CCL137 fibroblast cells. On the other hand, Silva et al. (8) have shown muscarinic receptor activity in purified clathrin-coated vesicles from bovine brain. Using electron microscopy, ␤ 2 -adrenorecep...

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This study examined the regulation of all known phosphodiesterase (PDE) type PDE4A, PDE4B and PDE4D splice variants in cortical neurons by cAMP signaling. Treatment with dibutyryl-cAMP (db-cAMP) caused the induction of two of the known splice variants, PDE4B2 and PDE4D1/PDE4D2. Although the splice variants PDE4A1, PDE4A5/PDE4A10, PDE4B3, PDE4B1, PDE4D3 and PDE4D4 were present in cortical neurons, their mRNA was not regulated at the transcriptional level by db-cAMP. To assess the increase in PDE4B2 and PDE4D1/D2 mRNA expression, the promoters containing these genes were characterized. Transcription from both promoters was stimulated by db-cAMP. Because chronic antidepressant treatment increases PDE4B, and not PDE4D, mRNA expression, we focused on the regulation of the PDE4B2 promoter by cAMP and CREB. Dominant negative mutants of CREB suppressed PDE4B2 promoter activity and a constitutively active form of CREB robustly stimulated it. These data demonstrate that in cortical neurons, a short PDE4B2 intronic promoter is regulated by CREB, confers cAMP responsitivity and directs PDE4B2 mRNA and protein expression.

Abstract:Previous evidence has suggested a role for calmodulin (CaM) in opioid receptor signaling. We demonstrate here that morphine stimulation of the -opioid (OP 3 ) receptor causes rapid CaM translocation to the nucleus in OP 3 -transfected human embryonic kidney (HEK)-293 cells and in SH-SY5Y human neuroblastoma cells. Ca 2ϩ influx into the cells resulting from OP 3 receptor activation was required for nuclear CaM translocation. Moreover, in HEK-OP 3 and SH-SY5Y cells, increased nuclear CaM content was associated with enhanced phosphorylation of the nuclear transcription factor cyclic AMP-responsive element-binding protein. This appeared to be mediated by Ca 2ϩ /CaM kinases and also by a pathway involving protein kinase C. CaM was previously shown to bind directly to the OP 3 receptor and to be released from the plasma membrane on agonist stimulation. To test whether OP 3 -mediated CaM release contributes to nuclear CaM signaling, we used a mutant OP 3 receptor (K273A) with reduced affinity for CaM that fails to release CaM from the plasma membrane. K273A-OP 3 activated Ca 2ϩ influx to a similar extent as wild-type OP 3 ; however, CaM translocation to the nucleus was attenuated. These results indicate that OP 3 -stimulated Ca 2ϩ influx results in nuclear CaM translocation, which appears to be enhanced by simultaneous CaM release by OP 3 wild-type receptor from plasma membranes. These results suggest a novel Ca 2ϩ /CaM signaling pathway of opioid receptors in the regulation of transcriptional activity.