Biomedical subjects

R R McKay

Publications and source records attributed to R R McKay.

10 recordsLinked to original sources

PubMed2000

Cloning and expression of the human transient receptor potential 4 (TRP4) gene: localization and functional expression of human TRP4 and TRP3.

Mammalian homologues of the Drosophila transient receptor potential (TRP) protein have been proposed to function as ion channels, and in some cases as store-operated or capacitative calcium entry channels. However, for each of the mammalian TRP proteins, different laboratories have reported distinct modes of cellular regulation. In the present study we describe the cloning and functional expression of the human form of TRP4 (hTRP4), and compare its activity with another well studied protein, hTRP3. When hTRP4 was transiently expressed in human embryonic kidney (HEK)-293 cells, basal bivalent cation permeability (barium) was increased. Whole-cell patch-clamp studies of hTRP4 expressed in Chinese hamster ovary cells revealed a constitutively active non-selective cation current which probably underlies the increased bivalent cation entry. Barium entry into hTRP4-transfected HEK-293 cells was not further increased by phospholipase C (PLC)-linked receptor activation, by intracellular calcium store depletion with thapsigargin, or by a synthetic diacylglycerol, 1-oleoyl-2-acetyl-sn-glycerol (OAG). In contrast, transient expression of hTRP3 resulted in a bivalent cation influx that was markedly increased by PLC-linked receptor activation and by OAG, but not by thapsigargin. Despite the apparent differences in regulation of these two putative channel proteins, green fluorescent protein fusions of both molecules localized similarly to the plasma-membrane, notably in discrete punctate regions suggestive of specialized signalling complexes. Our findings indicate that while both hTRP4 and hTRP3 can apparently function as cation channels, their putative roles as components of capacitative calcium entry channels are not readily demonstrable by examining their behaviour when exogenously expressed in cells.

Amino Acid Sequence↗

PubMed2000

Effects of elevated cytoplasmic calcium and protein kinase C on endoplasmic reticulum structure and function in HEK293 cells.

In human embryonic kidney (HEK) cells stably transfected with green fluorescent protein targeted to the endoplasmic reticulum (ER), elevation of intracellular Ca2+ ([Ca2+]i) altered ER morphology, making it appear punctate. Electron microscopy revealed that these punctate structures represented circular and branched rearrangements of the endoplasmic reticulum, but did not involve obvious swelling or pathological fragmentation. Activation of protein kinase C with phorbol 12-myristate 13-acetate (PMA), prevented the effects of ionomycin on ER structure without affecting the elevation of [Ca2+]i. These results suggest that protein kinase C activation alters cytoplasmic or ER components underlying the effects of high [Ca2+]i on ER structure. Treatment of HEK cells with PMA also reduced the size of the thapsigargin-sensitive Ca2+ pool and inhibited Ca2+ entry in response to thapsigargin. Thus, protein kinase C activation has multiple actions on the calcium storage and signalling function of the endoplasmic reticulum in HEK cells: (1) reduced intracellular Ca2+ storage capacity, (2) inhibition of capacitative Ca2+ entry, and (3) protection of the endoplasmic reticulum against the effects of high [Ca2+]i.

Calcium↗

PubMed1999

Capacitative calcium entry channels.

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.

Animals↗

PubMed1995

Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster.

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.

Alternative Splicing↗

PubMed1995

Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC), which enzymes play a pivotal role in one of the largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutant by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with norpA antiserum show that norpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutant exhibit very little to none. Immunohistochemical staining of tissue sections using norpA antiserum confirm that expression of norpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction.

Animals↗

PubMed1995

Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling.

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned from Drosophila melanogaster and one is from Artemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.

Animals↗

PubMed1995

A Drosophila gene that encodes a member of the protein disulfide isomerase/phospholipase C-alpha family.

Screening of a Drosophila genomic DNA library at reduced stringency hybridization conditions using a rat PLC alpha cDNA probe yielded a gene which encodes a member of the protein disulfide isomerase/PLC alpha family. The gene has been localized to band 74C on the left arm of the third chromosome and has been designated dpdi. Northern analysis shows that the dpdi gene encodes a transcript that is 2.3 kb in length and is present throughout development as well as in both heads and bodies of adults. The deduced dpdi protein is 496 amino acids in length and contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a sequence of four amino acids (KDEL) at the C-terminus which has been described by others as being responsible for retention of proteins in the endoplasmic reticulum. Overall, dpdi contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat PLC alpha (30% identical). However, it is unclear whether dpdi functions in vivo as a PDI or as a PLC, or both. Drosophila, with its well characterized genetics and the ability to generate mutants in a gene that has been cloned, provides an excellent system in which to resolve this issue.

Amino Acid Sequence↗

PubMed1994

Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA, as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.

Animals↗

PubMed1994

The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye.

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

Animals↗

PubMed1993

Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. Molecular cloning of the norpA gene revealed that it encodes phosphatidylinositol-specific phospholipase C, which enzymes play a pivotal role in one of the largest classes of signaling pathways known. We have used Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues to examine the tissue-specific expression of the norpA gene and found that it is expressed in a variety of tissues besides the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants, a result which is consistent with previous conclusions that the norpA gene encodes an essential component of the visual system. However, the norpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays which show that the norpA mutation reduces phospholipase C activity in each of the tissues in which norpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the norpA antiserum demonstrates that the norpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Since some of the above mentioned tissues that express norpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, we conclude that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction.

Animals↗