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...

The extracellular and intracellular domains of the human thyrotropin receptor were expressed in Escherichia coli and the proteins were used to produce monoclonal anti-receptor antibodies. Immunoblot studies and immunoaffinity purification showed that the receptor is composed of two subunits linked by disulfide bridges and probably derived by proteolytic cleavage of a single 90-kDa precursor. The extracellular a subunit (hormone binding) had an apparent molecular mass of 53 kDa (35 kDa after deglycosylation with N-glycosidase F). The membrane-spanning (3 subunit seemed heterogeneous and had an apparent molecular mass of 33-42 kDa. Human thyroid membranes contained a 2.5-to 3-fold excess of (3 subunits over a subunits. Immunocytochemistry showed the presence of both subunits in all the follicular thyroid cells, and both subunits were restricted to the basolateral region of the cell membrane.The thyrotropin receptor (TSHR) has been the subject of great interest for many years due to its physiological importance and its implication in Graves disease (1, 2). However, its rarity and fragility have precluded its purification, and indirect evidence has led to conflicting reports on its molecular structure (1-13). Total molecular masses of 90-500 kDa, with subunits varying in number from one to three and in mass from 15 to 90 kDa, have been reported. TSHR cDNAs have been cloned by cross-hybridization with related G-protein-coupled receptor cDNAs or by PCR amplification with homologous primers (14-17). The primary structure of the encoded protein (molecular mass, 84.5 kDa) has been deduced. The high sequence homology with the lutropin receptor, which is composed of a single polypeptide chain (18), led most researchers to hypothesize a similar structure for the TSHR.We have used Escherichia coli to express fragments corresponding to the extracellular and intracellular domains of the TSHR. Immunization of mice led to the production of monoclonal antibodies that were used for the immunochemical characterization ofthe receptor. Here we report evidence for the heterodimeric structure of the TSHR. MATERIALS AND METHODSPreparation of Anti-TSHR Monoclonal Antibodies. cDNA fragments encoding amino acids 19-243 or 604-764 of the human TSHR were introduced into the polylinker of the vector pUR292 (19) or pNMHUB (20). Fusion proteins of TSHR with f3-galactosidase and ubiquitin, respectively, were produced in E. coli. Cell lysates were prepared in buffer A (20 mM sodium phosphate/0.3 M NaCl/10 mM MgCl2/1% Triton X-100, pH 7.4) containing lysozyme at 5 mg/ml. After two freeze-thaw cycles, the lysate was treated with DNase I (0.1 mg/ml) at 20°C for 20 min. After centrifugation at 10,000x g for 30 min, the pellets were washed twice with buffer A containing 0.5% sodium deoxycholate and twice with 2 M guanidinium chloride and solubilized in 6 M guanidinium chloride/0.5 M dithiothreitol. Samples were dialyzed for 48 hr in 10 mM sodium phosphate/150 mM NaCl/8 M urea/10 mM dithiothreitol, pH 8.0. For immunization of mice, the samples ...

Capacitative Ca 2؉ entry (CCE) is Ca 2؉ entering after stimulation of inositol 1,4,5-trisphosphate (IP3) formation and initiation of Ca 2؉ store depletion. One hallmark of CCE is that it can also be triggered merely by store depletion, as occurs after inhibition of internal Ca 2؉ pumps with thapsigargin. Evidence has accumulated in support of a role of transient receptor potential (Trp) proteins as structural subunits of a class of Ca 2؉ -permeable cation channels activated by agonists that stimulate IP3 formation-very likely through a direct interaction between the IP3 receptor and a Trp subunit of the Ca 2؉ entry channel. The role of Trp's in Ca 2؉ entry triggered by store depletion alone is less clear. Only a few of the cloned Trp's appear to enhance this type of Ca 2؉ entry, and when they do, the effect requires special conditions to be observed, which native CCE does not. Here we report the full-length cDNA of mouse trp2, the homologue of the human trp2 pseudogene. Mouse Trp2 is shown to be readily activated not only after stimulation with an agonist but also by store depletion in the absence of an agonist. In contrast to other Trp proteins, Trp2-mediated Ca 2؉ entry activated by store depletion is seen under the same conditions that reveal endogenous store depletion-activated Ca 2؉ entry, i.e., classical CCE. The findings support the general hypothesis that Trp proteins are subunits of store-and receptor-operated Ca 2؉ channels.

The extracellular domain of the human FSH receptor was expressed in Escherichia coli as a fusion protein with ubiquitin. It was tagged with a poly-His tract which was used for its purification. Immunization of mice allowed the preparation of high affinity antireceptor monoclonal antibodies. The latter fell into two categories: some of them were inhibited hormone binding and adenylate cyclase activation whereas others were devoid of these properties. None of the antibodies had agonistic activity (i.e., stimulated adenylate cyclase). Immunoaffinity chromatography allowed us to purify the native receptor in a single step either from a permanently transfected L cell line (75% recovery) or from human ovaries (33% recovery). Immunoblotting of the receptor in human ovaries showed the presence of a major band of 87 kDa and of a minor band of 81 kDa. Endoglycosidase digestion and pulse-chase experiments showed the former to be the mature receptor and the latter the precursor containing mannose-rich carbohydrates. Thus, as in the case for the LH receptor, there was an accumulation (albeit to a lower degree) of the precursor in target cells. We did not detect variant forms of the protein corresponding to the alternative mRNA transcripts previously described. Additive binding to the receptor of several antibodies, but not of the same antibody, allowed us to establish a sandwich-type ELISA for the receptor (sensitivity approximately 1 fmol) and to obtain evidence against the existence of previously described oligomeric forms of the protein. All monoclonal antibodies were able to label the receptor immunocytochemically in transfected cells, and two of them were also able to detect it at the markedly lower physiological concentrations, i.e., in human Sertoli and granulosa cells.