Competitive coordination of the dual roles of the Hedgehog co-receptor in homophilic adhesion and signal reception

Abstract

Hedgehog (Hh) signaling patterns embryonic tissues and contributes to homeostasis in adults. In Drosophila, Hh transport and signaling are thought to occur along a specialized class of actin-rich filopodia, termed cytonemes. Here, we report that Interference hedgehog (Ihog) not only forms a Hh receptor complex with Patched to mediate intracellular signaling, but Ihog also engages in trans-homophilic binding leading to cytoneme stabilization in a manner independent of its role as the Hh receptor. Both functions of Ihog (trans-homophilic binding for cytoneme stabilization and Hh binding for ligand sensing) involve a heparin-binding site on the first fibronectin repeat of the extracellular domain. Thus, the Ihog-Ihog interaction and the Hh-Ihog interaction cannot occur simultaneously for a single Ihog molecule. By combining experimental data and mathematical modeling, we determined that Hh-Ihog heterophilic interaction dominates and Hh can disrupt and displace Ihog molecules involved in trans-homophilic binding. Consequently, we proposed that the weaker Ihog-Ihog trans interaction promotes and stabilizes direct membrane contacts along cytonemes and that, as the cytoneme encounters secreted Hh ligands, the ligands trigger release of Ihog from trans Ihog-Ihog complex enabling transport or internalization of the Hh ligand-Ihog-Patched -receptor complex. Thus, the seemingly incompatible functions of Ihog in homophilic adhesion and ligand binding cooperate to assist Hh transport and reception along the cytonemes.

Regulatory mechanisms of cytoneme-based morphogen transport

“…3a). In the latter study, homophilic Ihog interactions worked in trans to stabilize multiple discrete connections, indicating that enrichment of specific proteins in cytonemes can influence connection behavior [162].…”

“…Cytoneme contact has been observed in both fixed and live cellular imaging in fly and vertebrate models [67,104]. Live imaging studies reveal that cytoneme associations can occur as transient connections suggestive of scan and release activity, or stable connections that may be reinforced by adhesion protein or co-receptor functions [59,62,67,162] (Fig. 3).…”

“…4). Whereas live imaging of murine NIH3T3 cells overexpressing SHH and MYO10 revealed stable contact between cytoneme tips [67], analysis of Ihog-stabilized cytonemes in the Drosophila wing disc using GFP reconstitution across synaptic partners (GRASP) revealed multiple cytoneme-cytoneme contact points along the lengths of the extensions [162] (Fig. 3a).…”

Glypicans define unique roles for the Hedgehog co-receptors boi and ihog in cytoneme-mediated gradient formation

“…Second, we showed a different role of glypicans with respect to Boi: under the same experimental conditions, Boi plasma membrane levels are not affected, indicating that the modulation of Ihog by glypicans is specific. Third, we determined that the glypicans-Ihog interaction is mediated by both Ihog FNIII domains, Fn1 and Fn2; however, even though the Fn1 domain interacts equally with both glypicans (see also Yang et al, 2021), the Fn2 domain is almost dispensable for the interaction with Dlp, but necessary for that with Dally.…”

“…Our results support the hypothesis that the interactions of Hh, Ptc, and Ihog with the glypicans, among other complex components, might guide the Hh reception at cytoneme contact sites. In this context, it has recently been proposed that the Ihog-Ihog homophilic interaction is competing with the Ihog-Hh heterophilic interaction through the Ihog Fn1 domain (Yang et al, 2021). This competing interaction at the cytoneme contact sites between presenting and receiving cytonemes could allow the Hh reception.…”