Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.
The G protein-coupled receptor (GPCR) Smoothened (Smo) is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh) pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.
Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
Suresh
S
Marada
Department of Biochemistry and Molecular Biology, Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)University of Barcelona, Barcelona, Spain.
Gemma
G
Navarro
Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America; Summer Plus Program, Rhodes College, Memphis, Tennessee, United States of America.
Ashley
A
Truong
Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
Daniel P
DP
Stewart
Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
Angela M
AM
Arensdorf
Departments of Medicine and Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America.
Sigrid
S
Nachtergaele
Department of Biochemistry and Molecular Biology, Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)University of Barcelona, Barcelona, Spain.
Edgar
E
Angelats
Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
Joseph T
JT
Opferman
Departments of Medicine and Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America.
Rajat
R
Rohatgi
Department of Biochemistry and Molecular Biology, Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED)University of Barcelona, Barcelona, Spain; School of Pharmacy, University of East Anglia, Norwich, Norfolk, United Kingdom.
Peter J
PJ
McCormick
Department of Cell & Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
Stacey K
SK
Ogden
D000595:Amino Acid Sequence; D000818:Animals; D017124:Conserved Sequence; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction; D000072086:Smoothened Receptor; D013045:Species Specificity
D029721:Drosophila Proteins; D043562:Receptors, G-Protein-Coupled; D000072086:Smoothened Receptor; C100664:smo protein, Drosophila