Proteolytic processing of LRP2 on RPE cells regulates BMP activity to control eye size and refractive error

Abstract

Mutations in LRP2, a transmembrane receptor, cause ocular enlargement and high-myopia. LRP2 is expressed by the RPE and eye ciliary epithelia, binding many extracellular ligands, including Bmp4 and Shh. Signaling mediated by LRP2 is very context-dependent, and how multiple pathways are coordinated is unknown. Transcriptome analyses of ocular tissues revealed that controlled, sustained BMP signaling from the RPE is critical for normal eye growth and emmetropia (proper refraction). Using zebrafish, we demonstrate that BACE sheddase-dependent LRP2 cleavage produces a soluble domain that binds BMP4, inhibiting its signaling. We propose that controlled proteolytic cleavage of LRP2 makes two ligand-binding receptor forms available: a soluble BMP trap, and a membrane-bound RPE signaling facilitator. By modulating LRP2 cleavage, cells can fine-tune and coordinate multiple signaling pathways, as well as growth and turnover of the extracellular matrix, control of which is important to maintain proper eye size. This data supports the concept that LRP2 acts as a homeostasis node that buffers and integrates diverse signaling to regulate emmetropic eye growth.Author SummaryFor proper focusing and normal vision, the axial length of the eye needs to match the refractive power of the lens. This is achieved by fine-tuning multiple signaling pathways to regulate the shape of the eye primarily by remodeling of the sclera, the outermost layer of the eye. This process is termed emmetropization. Emmetropization cues are initiated by visual input, but how signals are transduced from the photoreceptors across the retinal pigment epithelium to the sclera is incompletely understood. Here we show that cleavage of Lrp2, a large receptor expressed on RPE cells in the eye, alters BMP signaling, which contributes to proper eye size control. Dysregulation of BMP signaling by a) absence of Lrp2 in mutant zebrafish or b) overexpression of BMP antagonists from the RPE both cause eye enlargement and myopia. Understanding how regulated cleavage of Lrp2 affects paracrine signaling provides critical insight to emmetropization, raising the possibility for development of therapeutic agents to combat the epidemic incidence of refractive error.

Precise Short Sequence Insertion in Zebrafish Using a CRISPR/Cas9 Approach to Generate a Constitutively Soluble Lrp2 Protein

“…Due to the similarities between lrp2 S4424N * /S4424N * and C23X/C23X homozygous zebrafish eyes, which share phenotypic changes including eye enlargement, myopia, and ciliary epithelial hypoplasia, it is likely that the mechanism that causes these phenotypes is the same. As detailed in our earlier work (Collery and Link, 2018), we propose that absence of Lrp2 protein or excessive levels of soluble Lrp2 domains have a similar effect on the eye, causing a reduction in proper emmetropic signaling. In both cases, membrane-bound Lrp2 is not present to facilitate normal regulation of eye size, and buphthalmia is the result.…”

“…Extracellular cleavage of LRP-1 protein has been well documented (Herz et al, 1988, 1990), and we have proposed that LRP2 is also cleaved to yield a soluble extracellular form, allowing LRP2 to act as a switch between membrane-bound endocytosis and soluble decoy (Collery and Link, 2018). Since the factor(s) that carry out cleavage of LRP2 are currently unknown, we elected to use CRISPR/Cas9 methods to generate a constitutively soluble version of Lrp2 in zebrafish by inserting a short cassette containing stop codons into the coding sequence just before the transmembrane domain.…”

“…Multiple Lrp2 mutant mouse lines have been generated that show enlarged eyes, but have inconsistencies in other phenotypes – one line shows high lethality while the other does not (Zarbalis et al, 2004; Spoelgen et al, 2005). We have recently demonstrated that extracellular cleavage of Lrp2 to release a ligand-binding domain may function as a switch, converting the membrane-bound endocytic receptor to a soluble decoy that alters signaling by bound ligands (Collery and Link, 2018). In the eye, soluble N-terminal domains of Lrp2 expressed from the RPE lead to eye enlargement and myopia similar to that seen in lrp2 mutant zebrafish.…”