Functional characterization of the three Drosophila retinal degeneration C (RDGC) protein phosphatase isoforms

Abstract

Drosophila retinal degeneration C (RDGC) is the founding member of the PPEF family of protein phosphatases. RDGC mediates dephosphorylation of the visual pigment rhodopsin and the TRP ion channel. From the rdgC locus, three protein isoforms, termed RDGC-S, -M, and -L, with different N-termini are generated. Due to fatty acylation, RDGC-M and -L are attached to the plasma membrane while RDGC-S is soluble. To assign physiological roles to these RDGC isoforms, we constructed flies that express various combinations of RDGC protein isoforms. Expression of the RDGC-L isoform alone did not fully prevent rhodopsin hyperphosphorylation and resulted in impaired photoreceptor physiology and in decelerated TRP dephosphorylation at Ser936. However, expression of RDGC-L alone as well as RDGC-S/M was sufficient to prevent degeneration of photoreceptor cells which is a hallmark of the rdgC null mutant. Membrane-attached RDGC-M displayed higher activity of TRP dephosphorylation than the soluble isoform RDGC-S. Taken together, in vivo activities of RDGC splice variants are controlled by their N-termini.

Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation

“…The rdgC gene encodes three RdgC isoforms (746, 677, and 661 aa) with varying N-terminal sequences (25). We chose the longest protein isoform as the reporter, which was incorporated with the mCherry tag at its C-terminus.…”

Differential activation of rhodopsin triggers distinct endocytic trafficking and recycling in vivo via differential phosphorylation

“…The rdgC gene encodes three RdgC isoforms (746, 677, and 661 aa) with varying N-terminal sequences [23]. We chose the longest protein isoform as the reporter, which was incorporated with the mCherry tag at its C-terminus.…”

The Role of Reversible Phosphorylation of Drosophila Rhodopsin

“…Strauch and colleagues found the two longer isoforms to be fatty acylated at the N-terminus, leading to their membrane association, whereas the shortest isoform was mainly cytoplasmic. Additionally, these three isoforms were demonstrated to serve a redundant phosphatase function regarding the dephosphorylation of rhodopsin albeit with varying physiological contributions [ 45 ]. Aside from rhodopsin, RDGC also dephosphorylates the Ca 2+ -dependent photoreceptor ion channel TRP at a serine residue at position 936 which results in a form of light adaptation that allows the processing of stimuli with a high-temporal resolution in bright conditions [ 46 ].…”

“…Based on a highly similar phenotype (which will be described in detail later) it has also been argued that mutants with a lack of Ca 2+ influx—specifically norpA (encoding PLCβ)—display the same excess of rhodopsin phosphorylation [ 61 , 94 ]. This hyperphosphorylation of rhodopsin’s C-terminus can be monitored by the inability of a monoclonal antibody to target the C-terminal epitope of rhodopsin (DSHB, 4C5), as evidenced by a total lack of signal in immunoblots of rdgC mutant flies after white illumination [ 45 , 95 ]. This phenomenon had originally been described in retinal tissue sections of blue-illuminated norpA mutants as “epitope masking” of the rhodopsin C-terminus by arrestin 2 [ 96 ].…”