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Interaction and colocalization of CaBP4 and Unc119 (MRG4) in photoreceptors.

ABSTRACT: To characterize the interaction of the neuron-specific protein CaBP4 with the synaptic photoreceptor protein Unc119 homolog (MRG4).The interaction of CaBP4 and Unc119 was studied using affinity chromatography, yeast two-hybrid system, coimmunoprecipitation, and gel overlay assay. The colocalization of CaBP4 and Unc119 was analyzed using immunohistochemistry. Unc119, CaBP4, and synaptic proteins were examined in photoreceptors using immunohistochemistry and in synaptic tangential sections of flatmounted frozen retinas using Western blot analysis.Biochemical evidence supported the interaction of CaBP4 with Unc119. CaBP4 and Unc119 colocalized in the photoreceptor synapse of adult retina and during postnatal retinal development. A reduction in Unc119 levels was observed in the photoreceptor terminals of CaBP4-knockout mice compared with wild-type mice and was higher than the reduction of other synaptic proteins.This study provides evidence for the interaction of CaBP4 with Unc119 at the photoreceptor synapse. This interaction suggests a functional relationship between CaBP4 and Unc119, further supporting a role for these proteins in neurotransmitter release and in the maintenance of the photoreceptor synapse.

SUBMITTER: Haeseleer F

PROVIDER: S-EPMC2670247 | biostudies-literature | 2008 Jun

REPOSITORIES: biostudies-literature

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Interaction and colocalization of CaBP4 and Unc119 (MRG4) in photoreceptors.

Haeseleer Françoise F

Investigative ophthalmology & visual science 20080222 6

<h4>Purpose</h4>To characterize the interaction of the neuron-specific protein CaBP4 with the synaptic photoreceptor protein Unc119 homolog (MRG4).<h4>Methods</h4>The interaction of CaBP4 and Unc119 was studied using affinity chromatography, yeast two-hybrid system, coimmunoprecipitation, and gel overlay assay. The colocalization of CaBP4 and Unc119 was analyzed using immunohistochemistry. Unc119, CaBP4, and synaptic proteins were examined in photoreceptors using immunohistochemistry and in syna ...[more]

PMID: 18296658

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Project description:Primary cilia are highly specialized small antenna-like cellular protrusions that extend from the cell surface of many eukaryotic cell types. The protein content inside cilia and cytoplasm is very different, but details of the sorting process are not understood for most ciliary proteins. Recently, we have shown that prenylated proteins are sorted according to their affinity to the carrier protein PDE6? and the ability of Arl3 but not Arl2 to release high affinity cargo inside the cilia (Fansa, E. K., Kösling, S. K., Zent, E., Wittinghofer, A., and Ismail, S. (2016) Nat. Commun. 7, 11366). Here we address the question whether a similar principle governs the transport of myristoylated cargo by the carrier proteins Unc119a and Unc119b. We thus analyzed the binding strength of N-terminal myristoylated cargo peptides (GNAT1, NPHP3, Cystin1, RP2, and Src) to Unc119a and Unc119b proteins. The affinity between myristoylated cargo and carrier protein, Unc119, varies between subnanomolar and micromolar. Peptides derived from ciliary localizing proteins (GNAT1, NPHP3, and Cystin1) bind with high affinity to Unc119 proteins, whereas a peptide derived from a non-ciliary localizing protein (Src) has low affinity. The peptide with intermediate affinity (RP2) is localized at the ciliary transition zone as a gate keeper. We show that the low affinity peptides are released by both Arl2·GppNHp and Arl3·GppNHp, whereas the high affinity peptides are exclusively released by only Arl3·GppNHp. Determination of the x-ray structure of myristoylated NPHP3 peptide in complex with Unc119a reveals the molecular details of high affinity binding and suggests the importance of the residues at the +2 and +3 positions relative to the myristoylated glycine for high and low affinities. The mutational analysis of swapping the residues at the +2 and +3 positions between high and low affinity peptides results in reversing their affinities for Unc119a and leads to a partial mislocalization of a low affinity mutant of NPHP3.

Dataset Information

Interaction and colocalization of CaBP4 and Unc119 (MRG4) in photoreceptors.

Publications

Interaction and colocalization of CaBP4 and Unc119 (MRG4) in photoreceptors.

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