Phototransduction in Drosophila Is Compromised by Gal4 Expression but not by InsP3 Receptor Knockdown or Mutation

We have previously shown that addition of Ins(1,3,4,5)P4 to permeabilized L1210 cells increases the amount of Ca2+ mobilized by a submaximal concentration of Ins(2,4,5)P3, and we suggested that, in doing this, Ins(1,3,4,5)P4 is not working via an InsP3 receptor but indirectly via an InsP4 receptor [Loomis-Husselbee, Cullen, Dreikhausen, Irvine and Dawson (1996) Biochem. J. 314, 811–816]. Here we have investigated whether this effect might be mediated by GAP1IP4BP, recently identified as a putative receptor for Ins(1,3,4,5)P4. GAP1IP4BP is a protein that interacts with one or more monomeric G-proteins, so we sought evidence for involvement of monomeric G-proteins in the effects of Ins(1,3,4,5)P4 in permeabilized L1210 cells. Guanosine 5´-[γ-thio]triphosphate (GTP[S]) enhanced the effect of Ins(1,3,4,5)P4 on Ins(2,4,5)P3-stimulated Ca2+ mobilization, but had no effect on the action of Ins(2,4,5)P3 alone. A specific enhancement of only the action of Ins(1,3,4,5)P4 was also seen with GTP[S]-loaded R-Ras or Rap1a (two G-proteins known to interact with GAP1IP4BP), whereas H-Ras was inactive at similar concentrations. Guanosine 5´-[β-thio]diphosphate (GDP[S]) did not alter the action of either Ins(2,4,5)P3 or Ins(1,3,4,5)P4. Finally, the addition of exogenous GAP1IP4BP, purified from platelets, markedly enhanced the effect of Ins(1,3,4,5)P4, and again, the amount of Ca2+ mobilized by Ins(2,4,5)P3 alone was unaltered. We conclude that the increase in Ins(2,4,5)P3-stimulated Ca2+ mobilization by Ins(1,3,4,5)P4 may be mediated by GAP1IP4BP or a closely related protein (such as GAP1m), and if so, the action of the GAP1 is not solely to regulate GTP loading of a G-protein, but rather it acts with a G-protein to cause its effect.

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Investigation of the roles of Ca2+ and InsP3 diffusion in the coordination of Ca2+ signals between connected hepatocytes

Journal of Cell Science ◽

10.1242/jcs.114.11.1999 ◽

2001 ◽

Vol 114 (11) ◽

pp. 1999-2007

Author(s):

Caroline Clair ◽

Cécile Chalumeau ◽

Thierry Tordjmann ◽

Josiane Poggioli ◽

Christophe Erneux ◽

...

Keyword(s):

Gap Junctions ◽

Gap Junction ◽

Significant Role ◽

Signaling Molecules ◽

Rat Hepatocyte ◽

Inositol 1,4,5 Trisphosphate ◽

Gap Junction Coupling ◽

Junction Coupling ◽

Insp3 Receptor

Glycogenolytic agonists induce coordinated Ca2+ oscillations in multicellular rat hepatocyte systems as well as in the intact liver. The coordination of intercellular Ca2+ signals requires functional gap-junction coupling. The mechanisms ensuring this coordination are not precisely known. We investigated possible roles of Ca2+ or inositol 1,4,5-trisphosphate (InsP3) as a coordinating messengers for Ca2+ spiking among connected hepatocytes. Application of ionomycin or of supra-maximal concentrations of agonists show that Ca2+ does not significantly diffuse between connected hepatocytes, although gap junctions ensure the passage of small signaling molecules, as demonstrated by FRAP experiments. By contrast, coordination of Ca2+ spiking among connected hepatocytes can be favored by a rise in the level of InsP3, via the increase of agonist concentrations, or by a shift in the affinity of InsP3 receptor for InsP3. In the same line, coordination cannot be achieved if the InsP3 is rapidly metabolized by InsP3-phosphatase in one cell of the multiplet. These results demonstrate that even if small amounts of Ca2+ diffuse across gap junctions, they most probably do not play a significant role in inducing a coordinated Ca2+ signal among connected hepatocytes. By contrast, coordination of Ca2+ oscillations is fully dependent on the diffusion of InsP3 between neighboring cells.

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Regulation of the Type III InsP3 Receptor by InsP3 and ATP

Biophysical Journal ◽

10.1016/s0006-3495(00)76289-8 ◽

2000 ◽

Vol 79 (1) ◽

pp. 271-278 ◽

Cited By ~ 38

Author(s):

Robert E. Hagar ◽

Barbara E. Ehrlich

Keyword(s):

Type Iii ◽

Insp3 Receptor

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Promoter elements determining weak expression of the GAL4 regulatory gene of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.8.4999-5009.1993 ◽

1993 ◽

Vol 13 (8) ◽

pp. 4999-5009

Author(s):

D W Griggs ◽

M Johnston

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Sequences ◽

Glucose Repression ◽

Transcriptional Start Site ◽

Regulatory Gene ◽

Distal Region ◽

Deletion Mapping ◽

Positive Elements ◽

Gal4 Expression ◽

Weak Expression

The GAL4 gene of Saccharomyces cerevisiae (encoding the activator of transcription of the GAL genes) is poorly expressed and is repressed during growth on glucose. To determine the basis for its weak expression and to identify DNA sequences recognized by proteins that activate transcription of a gene that itself encodes an activator of transcription, we have analyzed GAL4 promoter structure. We show that the GAL4 promoter is about 90-fold weaker than the strong GAL1 promoter and at least 7-fold weaker than the feeble URA3 promoter and that this low level of GAL4 expression is primarily due to a weak promoter. By deletion mapping, the GAL4 promoter can be divided into three functional regions. Two of these regions contain positive elements; a distal region termed the UASGAL4 (upstream activation sequence) contains redundant elements that increase promoter function, and a central region termed the UESGAL4 (upstream essential sequence) is essential for even basal levels of GAL4 expression. The third element, an upstream repression sequence, mediates glucose repression of GAL4 expression and is located between the UES and the transcriptional start site. The UASGAL4 is unusual because it is not interchangable with UAS elements in other yeast promoters; it does not function as a UAS element when inserted in a CYC1 promoter, and a normally strong UAS functions poorly in place of UASGAL4 in the GAL4 promoter. Similarly, the UES element of GAL4 does not function as a TATA element in a test promoter, and consensus TATA elements do not function in place of UES elements in the GAL4 promoter. These results suggest that GAL4 contains a weak TATA-less promoter and that the proteins regulating expression of this regulatory gene may be novel and context specific.

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GAL4–Expression System

Encyclopedic Reference of Genomics and Proteomics in Molecular Medicine ◽

10.1007/3-540-29623-9_7114 ◽

2006 ◽

pp. 635-635

Keyword(s):

Expression System ◽

Gal4 Expression

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Investigating the InsP3 Receptor in Living Cells by Caged InsP3

Methods in Molecular Biology - Inositol Phosphates ◽

10.1007/978-1-0716-0167-9_10 ◽

2019 ◽

pp. 121-129

Author(s):

Xin Hui ◽

Peter Lipp

Keyword(s):

Living Cells ◽

Insp3 Receptor

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Analysis of Gal4 Expression Patterns in Adult Drosophila Females

G3 Genes|Genome|Genetics ◽

10.1534/g3.120.401676 ◽

2020 ◽

Vol 10 (11) ◽

pp. 4147-4158

Author(s):

Lesley N. Weaver ◽

Tianlu Ma ◽

Daniela Drummond-Barbosa

Keyword(s):

Genetic Manipulation ◽

Expression Patterns ◽

Cell Types ◽

Tissue Expression ◽

Whole Body ◽

Specific Gene ◽

Specific Cell ◽

Gene Pathway ◽

Gal4 Expression ◽

Adult Drosophila

Precise genetic manipulation of specific cell types or tissues to pinpoint gene function requirement is a critical step in studies aimed at unraveling the intricacies of organismal physiology. Drosophila researchers heavily rely on the UAS/Gal4/Gal80 system for tissue-specific manipulations; however, it is often unclear whether the reported Gal4 expression patterns are indeed specific to the tissue of interest such that experimental results are not confounded by secondary sites of Gal4 expression. Here, we surveyed the expression patterns of commonly used Gal4 drivers in adult Drosophila female tissues under optimal conditions and found that multiple drivers have unreported secondary sites of expression beyond their published cell type/tissue expression pattern. These results underscore the importance of thoroughly characterizing Gal4 tools as part of a rigorous experimental design that avoids potential misinterpretation of results as we strive for understanding how the function of a specific gene/pathway in one tissue contributes to whole-body physiology.

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