Randall D. Shortridge

Two distantly positioned PDZ domains mediate multivalent INAD–phospholipase C interactions essential for G protein‐coupled signaling

Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein‐coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C‐β (PLC‐β), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C‐terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box‐homology region (a putative G protein‐interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD–NORPA interactions which are essential for G protein‐coupled phototransduction.

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

INOSITOL PHOSPHOLIPID AND INVERTEBRATE PHOTORECEPTORS

Phototransduction in vertebrate photoreceptors is now known to be mediated through a cascade of reactions in which photoexcitation of rhodopsin is coupled to the activation of cGMP phosphodiesterase through a class of guanine nucleotide binding protein (G protein)*, transducin, leading to the hydrolysis of cGMP to modulate the activity of cGMP-gated cation channels (reviewed by: Stryer, 1986; Pugh and Lamb, 1990). A substantial body of evidence has accumulated in recent years to suggest that an analogous process takes place in invertebrate photoreceptors involving the light-dependent activation of phospholipase C (PLC) as a primary effector enzyme. In close analogy with one of the most widely utilized signal generating mechanisms (Berridge, 1987), it is thought that, in this process, rhodopsin activates PLC through a G protein to hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP*) to generate diacylglycerol (DG) and inositol L4S-trisphosphate (IP3) (Model A of figure; Fein, 1986; Selinger and Minke, 1988). However, another line of evidence suggests that the generalized model for inositol lipid signaling may not be sufficient to explain invertebrate phototransduction. We review in this paper the current status of the field and alternative models for the inositol lipid cascade of invertebrate phototransduction. The first demonstration that the inositol lipid pathway may be involved in invertebrate phototransduction came from the injection of IP3 into Limulus ventral photoreceptors. IP3 produced bursts of transient depolarizations caused by membrane currents having the same reversal potential as those produced by light (Fein et al., 1984; Brown et a)., 1984b). Similarly, introduction of IP3 into housefly (Murca) photoreceptors by light-induced pinocytosis was found to cause a large increase in noisy depolarizations in the dark (Devary el al., 1987). Consistent with these findings, light-induced increases in IP3 content have been demonstrated in Limufus ventral photoreceptors (Brown et al., 1984b), squid retinae (Szuts et al., 1986; Brown et d., 1987; Baer and Saibil, 1988; Wood el al., 1989), and fly photoreceptors (Devary et al., 1987).

Membrane association of phospholipase c encoded by thenorpA gene ofdrosophila 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.

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

abstractPhosphatidylinositol-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 fromDrosophila melanogaster and one is fromArtemia, 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.

Substitution of a non-retinal phospholipase C in Drosophila phototransduction.

The Drosophila norpA gene encodes at least two subtypes of phospholipase C (PLC), one of which is essential for phototransduction and the other is utilized in signalling pathways other than phototransduction. The two subtypes of norpA‐PLC differ by 14 amino acids that have been proposed as important for the function of PLC in different signalling pathways. The present study aimed to determine whether norpA subtype II enzyme can functionally substitute for the subtype I enzyme in the phototransduction pathway. We found that the non‐retinal norpA‐PLC enzyme can substitute for its retinal counterpart, but that there is a reduced rate of repolarization of photoreceptors following intense light stimuli. This reduced repolarization might be due to the inability of a regulatory component being able to interact with the non‐retinal norpA‐PLC enzyme.

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.

Impact of Studies of the Drosophila norpA Mutation on Understanding Phototransduction

Abstract: Studies of the norpA mutation have significantly contributed to understanding the molecular and biochemical basis of phototransduction in Drosophila. Historical milestones in the study of the norpA mutation are reviewed and a contemporary model of the role of the NORPA protein in vision is presented. Questions awaiting further investigations are discussed.