During the last 2 years, our laboratory has worked on the elucidation of the molecular basis of capacitative calcium entry (CCE) into cells. Specifically, we tested the hypothesis that CCE channels are formed of subunits encoded in genes related to the Drosophila trp gene. The first step in this pursuit was to search for mammalian trp genes. We found not one but six mammalian genes and cloned several of their cDNAs, some in their full length. As assayed in mammalian cells, overexpression of some mammalian Trps increases CCE, while expression of partial trp cDNAs in antisense orientation can interfere with endogenous CCE. These findings provided a firm connection between CCE and mammalian Trps. This article reviews the known forms of CCE and highlights unanswered questions in our understanding of intracellular Ca 2؉ homeostasis and the physiological roles of CCE.The two primary second messengers mediating rapid responses of cells to hormones, autacoids, and neurotransmitters are cyclic nucleotides and Ca 2ϩ . Cyclic nucleotides act, for the most part, by activating protein kinases. The actions of Ca 2ϩ are more complex, in that this cation acts in two ways: directly, by binding to effector proteins, and indirectly, by first binding to regulatory proteins such as calmodulin, troponin C, and recoverin, which in turn associate and modulate effector proteins. Effector proteins regulated in these manners by Ca 2ϩ include not only protein kinases and protein phosphatases but also phospholipases and adenylyl cyclases, which are signaling enzymes in their own right, and an array of proteins involved in cellular responses that range from muscle contraction to glycogenolysis, endo-, exo-, and neurosecretion, cell differentiation, and programmed cell death. A common mechanism used by hormones and growth factors to signal through cytosolic Ca 2ϩ ([Ca 2ϩ ] i ) is activation of a rather complex reaction cascade that begins with stimulation of phosphoinositide-specific phospholipase C (PLC) enzymes, PLC␤ and PLC␥, and is followed sequentially by formation of diacylglycerol plus inositol 1,4,5-trisphosphate (IP3), liberation of Ca 2ϩ from intracellular stores, and finally, entry of Ca 2ϩ from the external milieu. The basic mechanisms used to signal through [Ca 2ϩ ] i are determined by the fact that the resting level of cytosolic Ca 2ϩ is very low, in the neighborhood of 100 nM, while that in intracellular stores and in the surrounding extracellular milieu is in the neighborhood of 2 mM, that is, Ϸ10,000-fold higher. As a result, [Ca 2ϩ ] i is set by the balance of two opposing forces. One is passive influx into the cytoplasm. It is driven by the electrochemical gradient and causes cytosolic [Ca 2ϩ ] i to rise without expenditure of energy. This influx is carefully controlled both at the level of the plasma membrane and at the level of the membranes, which delimit the internal storage compartment. Entry of Ca 2ϩ from the extracellular space occurs through three classes of Ca 2ϩ permeable gates: voltagedependent Ca 2ϩ...

Receptor recycling plays a critical role in the regulation of cellular responsiveness to environmental stimuli. Agonist-promoted phosphorylation of G protein-coupled receptors has been related to their desensitization, internalization, and sequestration. Dephosphorylation of internalized G protein-coupled receptors by cytoplasmic phosphatases has been shown to be pH-dependent, and it has been postulated to be necessary for receptors to recycle to the cell surface. The internalized V2 vasopressin receptor (V2R) expressed in HEK 293 cells is an exception to this hypothesis because it does not recycle to the plasma membrane for hours after removal of the ligand. Because this receptor is phosphorylated only by G protein-coupled receptor kinases (GRKs), the relationship between recycling and GRK-mediated phosphorylation was examined. A nonphosphorylated V2R, truncated upstream of the GRK phosphorylation sites, rapidly returned to the cell surface after removal of vasopressin. Less-drastic truncations of V2R revealed the presence of multiple phosphorylation sites and suggested a key role for a serine cluster present at the C terminus. Replacement of any one of Ser-362, Ser-363, or Ser-364 with Ala allowed quantitative recycling of full-length V2R without affecting the extent of internalization. Examination of the stability of phosphate groups incorporated into the recycling S363A mutant V2Rs revealed that the recycling receptor was dephosphorylated after hormone withdrawal, whereas the wild-type V2R was not, providing molecular evidence for the hypothesis that GRK sites must be dephosphorylated prior to receptor recycling. These experiments uncovered a role for GRK phosphorylation in intracellular sorting and revealed a GRK-dependent anchoring domain that blocks V2R recycling.The V1a and V2 vasopressin receptors (V1aR and V2R) are members of the G protein-coupled receptor family and both become phosphorylated upon activation by agonist (1, 2). For several receptors of this family phosphorylation and internalization have been shown to be a consequence of activation by ligand and seem to play a role in reducing the cellular responses to repeated exposure to hormones. Phosphorylation of G protein-coupled receptors enhances their ability to bind arrestin, which uncouples the receptors from G proteins, and helps recruit them to the clathrin-coated pits, which mediate the internalization process (3-7). Although phosphorylation and internalization of G protein-coupled receptors have been known to play a role in receptor desensitization for several years, the biochemical steps involved are not fully known and are still the subject of intense investigation.It has previously been observed that after removal of arginine vasopressin (AVP) from the medium and from the surface receptors with an acid wash, almost all of the internalized V1aR expressed in HEK 293 cells returns to the plasma membrane very rapidly (2), similar to what had been observed with isolated hepatocytes and smooth muscle vascular cells (8, 9). The human...

The V2 vasopressin receptor undergoes ligand-induced sequestration and desensitization (Birnbaumer, M., Antaramian, A., Themmen, A. P. N., and Gilbert, S. (1992) J. Biol. Chem. 267, 11783-11788). The V2 receptor expressed in transfected cells labeled with [32P] orthophosphate was phosphorylated following the addition of 100 nM arginine vasopressin (AVP). Phosphorylation was complete 5 min after addition of AVP, and was not stimulated by increased levels of Ca2+ or cAMP. The half-maximal dose of AVP that stimulated phosphorylation was 2.4 +/- 0.4 nM, similar to the receptor KD of 4. 5 +/- 0.4 nM. The role of phosphorylation on receptor desensitization was investigated by studying two vasopressin receptors 14 and 27 amino acids shorter than the wild type receptor. The missing segments were not needed for normal ligand binding or coupling to Gs, but the last 14 amino acids were required for phosphorylation. The truncated receptors exposed to 100 nM AVP were sequestered and desensitized. The R137H V2R mutant receptor that binds vasopressin with wild type-like affinity and does not couple to Gs (Rosenthal, W., Antaramian, A., Gilbert, S., and Birnbaumer, M. (1993) J. Biol. Chem. 268, 13030-13033) was phosphorylated and subjected to ligand-induced sequestration. These results established that phosphorylation is not essential for sequestration and desensitization of the V2 vasopressin receptor. Furthermore, they revealed that the conformation acquired after ligand occupancy is necessary for receptor phosphorylation and sequestration, while coupling to Gs is not.

The human V2 vasopressin receptor contains one consensus site for N-linked glycosylation at asparagine 22 in the predicted extracellular amino terminal segment of the protein. This segment also contains clusters of serines and threonines that are potential sites for O-glycosylation. Mutagenesis of asparagine 22 to glutamine abolished N-linked glycosylation of the V2 receptor (N22Q-V2R), without altering its function or level of expression. The N22Q-V2R expressed in transfected cells migrated in denaturing acrylamide gels as two protein bands with a difference of 7000 Da. Protein labeling experiments demonstrated that the faster band could be chase to the slower one suggesting the presence of O-linked sugars. Sialidase treatment of membranes from cells expressing the N22Q-V2R or of immunoprecipitated metabolically labeled V2R accelerated the migration of the protein in acrylamide gels demonstrating the existence of O-glycosylation, the first time this type of glycosylation has been found in a G protein coupled receptor. Synthesis of metabolically labeled receptor in the presence of 1 mM phenyl-N-acetyl-alpha-D-galactosaminide, a competitive inhibitor of N-acetyl-alpha-D-galactose and N-acetylneuraminic acid transferases, also produced a receptor that migrated faster in denaturing gels. Serines and threonines present in the amino terminus were analyzed by alanine scanning mutagenesis to identify the acceptor sites. O-glycosylation was found at most serines and threonines present in the amino terminus. Because the disappearance of a site opened the availability of others to the transferases, the exact identification of the acceptor sites was not feasible. The wild type V2R expressed in HEK 293, COS, or MDCK cells underwent N- and O-linked glycosylation. The mutant V2R bearing all serine/threonine substitutions by alanine at the amino terminus yielded a receptor functionally indistinguishable from the wild type protein, whose mobility in polyacrylamide gels was no longer affected by sialidase treatment.

Palmitoylation of the V2 vasopressin receptor (V2R) and its functional role were investigated in transfected cells. Palmitoylation was assessed by incubating transfected cells with [3H]palmitic acid and immunoprecipitating the receptor with an antibody raised against a portion of the third intracellular loop of V2R. Wild-type and nonglycosylated V2R yielded tritium signals at 45-55 and 40 kDa, respectively, demonstrating that the V2R is palmitoylated and that receptor palmitoylation is independent of glycosylation. Substitution of CC341/342 for serines eliminated receptor palmitoylation, whereas replacement of a single amino acid, C341S or C342S, restored partial palmitoylation. Saturation binding assays revealed decreased cell surface expression of the nonpalmitoylated receptor compared with the wild-type; this effect was more pronounced when a truncated form of V2R (G345ter) was studied. The presence of either cysteine residue (C341S or C342S) elevated receptor expression to normal levels, most likely due to the partial restoration of palmitoylation. Ligand binding affinity, hormone-induced stimulation of adenylyl cyclase activity, receptor internalization, and desensitization were not affected by the absence of palmitoylation. No increase but rather a slight decrease in the extent of receptor palmitoylation was detected after exposure to vasopressin. It was concluded that the V2R is palmitoylated in both cysteines, each cysteine is palmitoylated independently from the other, and palmitoylation enhances cell surface expression of the V2R.

The V1a arginine vasopressin receptor (V1aR) expressed in HEK 293 cells was phosphorylated after binding to arginine vasopressin (AVP). The phosphate was incorporated very rapidly into the protein but remained attached for a very short time despite the continuous presence of hormone. The extent of phosphorylation depended upon the concentration of AVP suggesting the involvement of G-protein-coupled receptor kinases. Protein kinase C (PKC) contributed to V1aR phosphorylation as demonstrated by the fact that inhibition of the kinase decreased the amount of phosphate incorporated into the receptor. However, PKC activity was not responsible for the transient nature of V1aR phosphorylation. The hormone-free receptor could be phosphorylated by phorbol ester-activated PKC. Although the phosphorylation was transient, the phosphate groups incorporated remained on the receptor protein longer than those incorporated after AVP treatment. PKC phosphorylation of unoccupied V1aR was not sufficient to promote sequestration. Vasopressin also promoted sequestration of about 80% of the surface receptor, but measurements of the rate of accumulation of inositol phosphates in the sustained presence of the ligand did not reveal a significant desensitization of coupling to phospholipase C activity. The addition of a V1aR antagonist inhibited the sustained accumulation of inositol phosphates establishing that the sustained stimulation of PLC was mediated by receptors located on the cell surface. The transient character of V1aR phosphorylation seemed intrinsic to the receptor protein rather than a consequence of signaling within the cell, and receptor sequestration appeared to be responsible for the desensitization observed in HEK 293 cells.Agonist binding and activation of many G-protein-coupled receptors is followed by desensitization, an attenuation of the cellular response to the ligand that prevents sustained stimulation. Studies with rhodopsin and the ␤2-adrenergic receptor have implicated phosphorylation in receptor desensitization (1, 2) Receptor phosphorylation promotes the binding of arrestin, which in turn uncouples the receptor from the G-protein and enhances sequestration. In the particular case of the ␤2-adrenergic receptor, it has been postulated that sequestration of the phosphorylated receptor facilitates cleavage of the phosphate groups by phosphatases that associate with the endosomes and allows the recycling of the de-phosphorylated active protein to the cell surface (3). Data obtained with other G-protein-coupled receptors have indicated that phosphorylation is not required to observe receptor sequestration and desensitization. For example, a truncated form of the V2 vasopressin receptor lacking the phosphorylation acceptor sites at the carboxyl-terminal end was sequestered and desensitized, although to a lesser extent than the wild type receptor (4). Similarly, a mutant angiotensin II receptor missing a portion of the carboxyl terminus did not exhibit ligand-induced sequestration although it contained a putative GRK...

Gene therapy has been considered a strategy for delivery of therapeutic nucleic acids to a specific site. Calcium phosphates are one gene delivery vector group of interest. However, low transfection efficiency has limited the use of calcium phosphate in gene delivery applications. Present work aims at studying the fabrication of strontium substituted calcium phosphate nanoparticles with improved gene delivery related properties. Strontium substituted calcium phosphate was prepared using a simple sol gel method. X-ray diffraction analysis, Fourier transform infrared spectroscopy, transmission electron microscopy, specific surface area analysis, zeta potential measurement and ion release evaluation were used to characterize the samples. This characterization showed strontium and carbonate co-substituted calcium phosphate which resulted in nano size particles with low crystallinity, high specific surface area, positive surface charge, and a high dissolution rate. These improved properties could increase the DNA concentration on the vector as well as the endosomal escape of the complex that leads to higher transfection efficiency of this novel gene delivery vector.

Arrestins have been shown to facilitate the recruitment of G protein-coupled receptors to the clathrin-coated vesicles that mediate their internalization. After 8 Arg-vasopressin-induced internalization, the human V2 vasopressin receptor failed to recycle to the cell surface, whereas the vasopressin type 1a receptor (V1a) subtype did. The possibility that the lack of recycling could identify a novel role for arrestins was investigated by examining the effect of coexpressing wild-type and dominant negative arrestins on the recycling of wild-type and mutant V2 and V1a receptors. Coexpression of the V1a or V2 receptors with the last 100 amino acids of arrestin reduced significantly their internalization, whereas coexpression of wildtype and mutant arrestins had diverse effects on internalization.Arrestin3 but not arrestin2 increased the internalization of the V1aR without altering its recycling pattern. Both nonvisual arrestins enhanced vasopressin type 2 receptor (V2R) internalization, inducing the appearance of a pool of recycling receptor in addition to the nonrecycling pool. The effect of arrestins on the internalization of the chimeric V1a/V2 receptor and its reciprocal chimera was specified by the identity of the carboxylterminal segment. The S363A mutation that confers recycling to the V2R did not alter its interaction with arrestins. Truncation of the carboxyl-terminal segment of the V2R impaired ligandinduced internalization that could be fully restored by wild-type arrestins. Internalization of the V2 and V1a receptors required dynamin GTPase activity.