The protein-specific activities of the transmembrane modules of Ptch1 and Ptch2 are determined by their adjacent protein domains

Andrew J Fleet,Paul A Hamel

doi:10.1074/jbc.ra118.004478

Abstract

Signaling through the Hedgehog (Hh) pathway is mediated by the Patched (Ptch) family of proteins. Although the vertebrate Ptch proteins Ptch1 and Ptch2 harbor two closely related transmembrane modules related to sterol-sensing domains (SSDs), the role of these closely related receptors in the Hh pathway are not equivalent. Ptch1 is essential for development and appears to be the principal receptor mediating responses to Hh ligands, whereas Ptch2 is nonessential, and its role in Hh-signaling remains ambiguous. We hypothesized that the SSDs of the Ptch proteins function as generic modules whose protein-specific activities are determined by the adjacent cytoplasmic and luminal domains. We first showed that individual N-terminal and C-terminal halves of Ptch1 associated noncovalently to mediate ligand-dependent regulation of Hh signaling. The analogous regions of Ptch2 also interacted noncovalently but did not repress the Hh pathway. However, the SSD of Ptch2 were capable of repressing Hh signaling, as determined using chimeric proteins where the SSDs of Ptch1 were replaced by those from Ptch2. Replacement of the SSDs of Ptch1 with the analogous regions from the cholesterol transporter NPC1 failed to produce a chimeric protein capable of Hh repression. Further refinement of the specific regions in Ptch1 and Ptch2 revealed that specific cytoplasmic domains of Ptch1 were necessary but not sufficient for repression of Hh signaling and that the two principal luminal domains of Ptch1 and Ptch2 were interchangeable. These data support a model where the SSDs of the Ptch family proteins exhibit generic activities and that the adjacent cytoplasmic and luminal domains determine their protein-specific activities.

Highlights

Signaling through the Hedgehog (Hh) pathway is mediated by the Patched (Ptch) family of proteins
The luminal regions give rise to a predicted structure similar to the luminal loops in Niemann–Pick disease type C1 (NPC1). Despite these predicted similarities and the recent evidence that loss of NPC1 may alter cilium-dependent Hh signaling activities [34, 35], NPC1 does not appear to modulate Hh signaling directly [36]. These data suggest that the transmembrane modules of Ptch1 and NPC1 may harbor distinct transport activities either because of intrinsic differences in these modules or to protein-specific activities imparted by their respective adjacent domains
We addressed whether the transmembrane domains, referred to here as SSD1 and SSD2, of Ptch1, Ptch2, and NPC1 behave as generic transmembrane modules whose protein-specific activities are determined by the adjacent luminal and cytoplasmic domains

Summary

The abbreviations used are

Hedgehog; Ptch, Patched; RND, resistance– nodulation– division; SSD, sterol-sensing domain; aa, amino acids; HA, hemagglutinin; ML, middle loop; HEK, human embryonic kidney; MEF, mouse embryonic fibroblast; SH, Src homology; gRNA, guide RNA; Endo H, endoglycosidase H; PNGase F, peptide:N-glycosidase F. The structures of the luminal and transmembrane domains for human and mouse Ptch, alone or in complex with Shh-ligand, were solved using cryo-EM (30 –32) These structures revealed that the two luminal domains and the two SSD-like regions, respectively, form distinct but closely apposed modules. Despite these predicted similarities and the recent evidence that loss of NPC1 may alter cilium-dependent Hh signaling activities [34, 35], NPC1 does not appear to modulate Hh signaling directly [36] These data suggest that the transmembrane modules of Ptch and NPC1 may harbor distinct transport activities either because of intrinsic differences in these modules or to protein-specific activities imparted by their respective adjacent domains. We further showed that the generic activities of the SSD could not be extended to NPC1 because replacement of either of these domains in Ptch with the analogous regions of NPC1 generated proteins incapable of regulating the Hh signaling pathway

Results

Discussion

Experimental procedures