A Highly Conserved Amino-terminal Region of Sonic Hedgehog Is Required for the Formation of Its Freely Diffusible Multimeric Form

Abstract

Although members of the Hedgehog (Hh) family were initially described as morphogens, many of these early conclusions were based on experiments that used non-physiologically relevant forms of Hh. Native Hh is modified by cholesterol (HhNp) and palmitate. These hydrophobic modifications are responsible for the ability of Hh to associate with cellular membranes, a property that initially appeared inconsistent with its ability to act far from its site of synthesis. Although it is now clear that Hh family members are capable of acting directly in long-range signaling, the form of Hh capable of this activity remains controversial. We have previously provided evidence for a freely diffusible multimeric form of Sonic Hedgehog (Shh) termed s-ShhNp, which is capable of accumulating in a gradient fashion through a morphogenic field. Here, we provide further evidence that s-ShhNp is the physiologically relevant form of Shh. We show that the biological activity of freely diffusible ShhNp resides in its multimeric form and that this multimeric form is exceedingly stable, even to high concentrations of salt and detergent. Furthermore, we now validate the Shh-Shh interactions previously observed in the crystal structure of human Shh, showing that a highly conserved amino-terminal domain of Shh is important for the formation of s-ShhNp. We also conclusively show that palmitoylation is required for s-ShhNp formation. Thus, our results identify both protein-protein and protein-lipid interactions that are required for s-ShhNp formation, and provide the first structural analyses supporting the existence of Shh multimers. Although members of the Hedgehog (Hh) family were initially described as morphogens, many of these early conclusions were based on experiments that used non-physiologically relevant forms of Hh. Native Hh is modified by cholesterol (HhNp) and palmitate. These hydrophobic modifications are responsible for the ability of Hh to associate with cellular membranes, a property that initially appeared inconsistent with its ability to act far from its site of synthesis. Although it is now clear that Hh family members are capable of acting directly in long-range signaling, the form of Hh capable of this activity remains controversial. We have previously provided evidence for a freely diffusible multimeric form of Sonic Hedgehog (Shh) termed s-ShhNp, which is capable of accumulating in a gradient fashion through a morphogenic field. Here, we provide further evidence that s-ShhNp is the physiologically relevant form of Shh. We show that the biological activity of freely diffusible ShhNp resides in its multimeric form and that this multimeric form is exceedingly stable, even to high concentrations of salt and detergent. Furthermore, we now validate the Shh-Shh interactions previously observed in the crystal structure of human Shh, showing that a highly conserved amino-terminal domain of Shh is important for the formation of s-ShhNp. We also conclusively show that palmitoylation is required for s-ShhNp formation. Thus, our results identify both protein-protein and protein-lipid interactions that are required for s-ShhNp formation, and provide the first structural analyses supporting the existence of Shh multimers. The Hedgehog (Hh) 3The abbreviations used are: Hh, Hedgehog; Shh, Sonic Hedgehog; HhN, amino-terminal domain of Hh; HhC, carboxyl-terminal domain of Hh; HhNp, HhN post-translationally modified by the covalent addition of cholesterol; ShhN, recombinant form of Shh; wt, wild type; DMEM, Dulbecco's modified Eagle's medium. family of proteins plays an important instructive role in metazoan development (1.Ingham P.W. McMahon A.P. Genes Dev. 2001; 15: 3059-3087Crossref PubMed Scopus (2532) Google Scholar). More recently, the human Hh family members Sonic Hedgehog (Shh) and Indian Hedgehog were shown to play an important role as mitogens and survival factors for the growth of numerous types of tumor cells (2.Robbins D.J. Goetz J.A. Yuan Z. Stegman M.A. Curr. Cancer Ther. Rev. 2005; 1: 227-288Crossref Google Scholar, 3.Beachy P.A. Karhadkar S.S. Berman D.M. Nature. 2004; 432: 324-331Crossref PubMed Scopus (1032) Google Scholar, 4.Sanchez P. Clement V. Ruiz i Altaba A. Cancer Res. 2005; 65: 2990-2992Crossref PubMed Scopus (78) Google Scholar). Although the physiological responses and signaling pathway of Hh are well studied, less is known about how Hh family members function as biological ligands. Questions as to the state of the physiologically relevant ligand, how the ligand moves through extracellular space, and even how it exits cells to reach the plasma membrane, remain unanswered (5.Goetz J.A. Suber L.M. Zeng X. Robbins D.J. BioEssays. 2002; 24: 157-165Crossref PubMed Scopus (52) Google Scholar, 6.Torroja C. Gorfinkiel N. Guerrero I. J. Neurobiol. 2005; 64: 334-356Crossref PubMed Scopus (68) Google Scholar). One reason for these remaining questions is the unusual biochemistry of the Hh family of proteins. Hh is synthesized as a protein of ∼45 kDa, which subsequently undergoes an intramolecular proteolytic cleavage into an amino-terminal domain (HhN) and a carboxyl-terminal domain (HhC) (7.Lee J.J. Ekker S.C. von Kessler D.P. Porter J.A. Sun B.I. Beachy P.A. Science. 1994; 266: 1528-1537Crossref PubMed Scopus (457) Google Scholar, 8.Bumcrot D.A. Takada R. McMahon A.P. Mol. Cell Biol. 1995; 15: 2294-2303Crossref PubMed Scopus (262) Google Scholar). During this cleavage, the amino-terminal domain is post-translationally modified by the covalent addition of cholesterol (HhNp), with the carboxyl-terminal domain acting as a cholesterol transferase (9.Porter J.A. Young K.E. Beachy P.A. Science. 1996; 274: 255-259Crossref PubMed Scopus (1126) Google Scholar). HhNp is sufficient to activate all known Hh-dependent processes (10.Lai C.J. Ekker S.C. Beachy P.A. Moon R.T. Development. 1995; 121: 2349-2360PubMed Google Scholar, 11.Roelink H. Augsburger A. Heemskerk J. Korzh V. Norlin S. Ruiz i Altaba A. Tanabe Y. Placzek M. Edlund T. Jessell T.M. Dodd J. Cell. 1994; 76: 761-775Abstract Full Text PDF PubMed Scopus (762) Google Scholar, 12.Fietz M.J. Jacinto A. Taylor A.M. Alexandre C. Ingham P.W. Curr. Biol. 1995; 5: 643-650Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13.Porter J.A. von Kessler D.P. Ekker S.C. Young K.E. Lee J.J. Moses K. Beachy P.A. Nature. 1995; 374: 363-366Crossref PubMed Scopus (442) Google Scholar, 14.Marti E. Bumcrot D.A. Takada R. McMahon A.P. Nature. 1995; 375: 322-325Crossref PubMed Scopus (445) Google Scholar, 15.Fan C.M. Porter J.A. Chiang C. Chang D.T. Beachy P.A. Tessier-Lavigne M. Cell. 1995; 81: 457-465Abstract Full Text PDF PubMed Scopus (317) Google Scholar). HhNp is also modified by the covalent addition of a second lipid, palmitate, to its extreme amino terminus (16.Pepinsky R.B. Zeng C. Wen D. Rayhorn P. Baker D.P. Williams K.P. Bixler S.A. Ambrose C.M. Garber E.A. Miatkowski K. Taylor F.R. Wang E.A. Galdes A. J. Biol. Chem. 1998; 273: 14037-14045Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). This second lipid modification is added to Hh by a protein termed Skinny Hedgehog (Skn) (17.Micchelli C.A. The I. Selva E. Mogila V. Perrimon N. Development. 2002; 129: 843-851PubMed Google Scholar, 18.Lee J.D. Treisman J.E. Curr. Biol. 2001; 11: 1147-1152Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 19.Chamoun Z. Mann R.K. Nellen D. von Kessler D.P. Bellotto M. Beachy P.A. Basler K. Science. 2001; 293: 2080-2084Crossref PubMed Scopus (349) Google Scholar, 20.Amanai K. Jiang J. Development. 2001; 128: 5119-5127PubMed Google Scholar), which has homology to a family of O-acyl transferases. The exact functions of these lipid modifications to HhN remain uncertain. However, it has been suggested that cholesterol plays a role in localizing Hh to specific micro-domains on the plasma membrane (21.Rietveld A. Neutz S. Simons K. Eaton S. J. Biol. Chem. 1999; 274: 12049-12054Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar), whereas the palmitate on Hh may play a role in increasing its potency (22.Taylor F.R. Wen D. Garber E.A. Carmillo A.N. Baker D.P. Arduini R.M. Williams K.P. Weinreb P.H. Rayhorn P. Hronowski X. Whitty A. Day E.S. Boriack-Sjodin A. Shapiro R.I. Galdes A. Pepinsky R.B. Biochemistry. 2001; 40: 4359-4371Crossref PubMed Scopus (132) Google Scholar) or movement (23.Chen M.H. Li Y.J. Kawakami T. Xu S.M. Chuang P.T. Genes Dev. 2004; 18: 641-659Crossref PubMed Scopus (293) Google Scholar). The hydrophobic nature of HhNp makes studying its biochemistry difficult. Thus, the initial of Hh family members a recombinant form of Shh that which many of the biological to (10.Lai C.J. Ekker S.C. Beachy P.A. Moon R.T. Development. 1995; 121: 2349-2360PubMed Google Scholar, 12.Fietz M.J. Jacinto A. Taylor A.M. Alexandre C. Ingham P.W. Curr. Biol. 1995; 5: 643-650Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 13.Porter J.A. von Kessler D.P. Ekker S.C. Young K.E. Lee J.J. Moses K. Beachy P.A. Nature. 1995; 374: 363-366Crossref PubMed Scopus (442) Google Scholar, 14.Marti E. Bumcrot D.A. Takada R. McMahon A.P. Nature. 1995; 375: 322-325Crossref PubMed Scopus (445) Google Scholar, C.M. Tessier-Lavigne M. Cell. 1994; Full Text PDF PubMed Scopus Google Scholar, H. Porter J.A. Chiang C. Tanabe Y. Chang D.T. Beachy P.A. Jessell T.M. Cell. 1995; 81: Full Text PDF PubMed Scopus Google Scholar). However, the potency of recombinant less that of which been from the of cells Shh (16.Pepinsky R.B. Zeng C. Wen D. Rayhorn P. Baker D.P. Williams K.P. Bixler S.A. Ambrose C.M. Garber E.A. Miatkowski K. Taylor F.R. Wang E.A. Galdes A. J. Biol. Chem. 1998; 273: 14037-14045Abstract Full Text Full Text PDF PubMed Scopus (599) Google Scholar). Although ShhNp ShhN, it initially to freely a for ShhNp to act as a J.A. Ekker S.C. von Kessler D.P. Young K.E. Y. Beachy P.A. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). We have described a freely diffusible form of Shh we s-ShhNp, which as a of This form of ShhNp to Shh with an form of Shh X. Goetz J.A. Suber L.M. C.M. Robbins D.J. Nature. 2001; PubMed Scopus Google Scholar). is to form that formation or both of its lipid We suggested that s-ShhNp is the physiologically relevant form of Shh, a gradient of this form of Shh of Shh. The exact of Shh in s-ShhNp is as is the of factors that may act as the Shh More recently, it has been suggested that palmitoylation of ShhNp may required for (23.Chen M.H. Li Y.J. Kawakami T. Xu S.M. Chuang P.T. Genes Dev. 2004; 18: 641-659Crossref PubMed Scopus (293) Google Scholar). However, it has also been that s-ShhNp may a activity form ShhNp and that palmitoylation is required for J. T. Lee J.D. Development. 2004; PubMed Scopus (68) Google Scholar). these analyses of s-ShhNp, a of s-ShhNp has been The crystal structure of human recombinant protein is to that of T.M. Porter J.A. Beachy P.A. D.J. Nature. 1995; PubMed Scopus Google Scholar, R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). However, the structure of human from that of in the structure of a highly conserved amino-terminal domain in human This amino-terminal domain of is the highly conserved of Hh family to this the amino terminus of from the of the it appeared to by interactions with Although it has been suggested that the palmitoylation of ShhNp is required to form s-ShhNp, the human in this crystal structure the that the Shh-Shh crystal observed in the human structure in s-ShhNp, we amino in this amino-terminal domain of Shh to a structure function analyses of s-ShhNp. Here, we provide evidence that of s-ShhNp from a Shh the of an This s-ShhNp is highly and stable, remaining multimeric in the of high concentrations of salt and detergent. this of s-ShhNp, we show that a of protein-protein as well as protein-lipid interactions are required for the formation of s-ShhNp. Furthermore, our results provide the first evidence based on the structure of Shh for the existence of Shh supporting the biological of of the amino-terminal domain of in a of Shh Shh and Shh were into a H. Augsburger A. Heemskerk J. Korzh V. Norlin S. Ruiz i Altaba A. Tanabe Y. Placzek M. Edlund T. Jessell T.M. Dodd J. Cell. 1994; 76: 761-775Abstract Full Text PDF PubMed Scopus (762) Google Scholar). The of Shh were by its the as the The were used in the which amino which amino which amino which to which to which to which to which to which to which to which to and which to The of the Shh by we the Shh amino from or Shh and of Shh were into cells as the cells were with by the addition of The cells were and for as described D.J. K.E. R. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The of these cells and to clear cellular in for of the were directly by and for the of on or by to its multimeric state activity were in and a of The activity for all the is as of Shh and for The a This for and a as previously described X. Goetz J.A. Suber L.M. C.M. Robbins D.J. Nature. 2001; PubMed Scopus Google Scholar). The used to the were kDa, and The in with were and the Shh by with the Shh is a the ability of the to a Shh and the ability of that to to N. T. Wang Porter J.A. T.M. D.J. Beachy P.A. S. A. 1999; PubMed Scopus Google Scholar). all the were by the we that structure is to analyses of the cells were with of in the of to ability to to Shh in the of detergent. that cells are by concentrations we of Shh on a in and were and with to a of for Shh activity cells as previously described J. Wang Mann R.K. Beachy P.A. Nature. PubMed Scopus Google Scholar). and of of s-ShhNp has been Here, we the and activity of s-ShhNp a of that is required to this freely diffusible form of ShhNp to we a that has been to of ShhNp the of an as a a of s-ShhNp cells were with concentrations of the to activate Shh from these cells and used to the and activity of Shh. Shh in in a to Although Shh in the of its addition ShhNp in The ShhNp in the showing a an J. Wang Mann R.K. Beachy P.A. Nature. PubMed Scopus Google Scholar). We the from cells on a to that the s-ShhNp. were the Shh by and for Shh. ShhNp from the of cells in a to that previously described for s-ShhNp, with the of Shh as a with a of of ShhNp also observed in with a with it The of multimeric to ShhNp in the by cells We the ability of s-ShhNp to high salt or high to the of s-ShhNp the high salt both the and the were to these s-ShhNp through the in a to that of s-ShhNp with a and a the of s-ShhNp in a we of in the of these the of Shh in the as a with the s-ShhNp s-ShhNp Thus, cells of s-ShhNp in a form that is to both high concentrations of salt and detergent. Although we have previously shown that ShhNp in is it remains as to this activity to s-ShhNp or the form of ShhNp in the is the of this this we from the cells and it a with a of with The were with an of for a of and added to cells to for Shh these the of Shh activity from the in a that well with Shh with a and a Thus, the activity of ShhNp in resides in the Shh that s-ShhNp the of activity in Shh of Shh have provided results on the role palmitoylation plays in s-ShhNp formation (23.Chen M.H. Li Y.J. Kawakami T. Xu S.M. Chuang P.T. Genes Dev. 2004; 18: 641-659Crossref PubMed Scopus (293) Google Scholar, J. T. Lee J.D. Development. 2004; PubMed Scopus (68) Google Scholar). we to the role the amino-terminal domain of Shh plays in its and this the site of we our analyses of s-ShhNp by the palmitate site and of these were to cholesterol modified forms in a to Shh These were also highly in Shh, with palmitoylation a role in Shh to cellular Furthermore, from cells these Shh less activity that of Shh with palmitoylation of an important role in Shh of and that these proteins in a to ShhN, as a form These results are with palmitoylation of Shh an important role in the formation of s-ShhNp. of Shh the crystal structure of specific amino that appeared to the of Shh R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). These amino and on its amino-terminal domain as well as a of amino the the role these amino play in Shh and activity we in Shh, and to the of the amino that these Shh or Shh, were into and the were for Shh or multimeric state We also the from these which that all the proteins to as Shh, for both and forms of Shh The of Shh in of or cells to that of Shh The activity of these Shh well with Shh protein activity to Shh, activity to that of from and activity that on to that of Shh, that these have on the or activity of Shh. on these results we an and the highly conserved to an the of this conserved amino this Shh from our crystal as the of to from its form to ShhNp in the of cells We from cells the Shh a to directly the ability of these Shh crystal site to form s-ShhNp. The ability of and to form to Shh. The of multimeric to ShhNp is for Shh, whereas it is less for these We were to in the of cells it to a of its The of these Shh to form s-ShhNp However, we observed in the of the for from an ShhNp to We the reason for this that it the ability of these to form Shh multimers. These results are with the role the amino-terminal domain of Shh plays in activity and of amino important for s-ShhNp formation. The of cells Shh, or a were by for the of Shh the form of Shh or Shh in the of these cells by for its ability to activate a Hh-dependent or a to the of s-ShhNp. The in which are the of Shh, and in the that observed in the the human crystal structure on the amino-terminal domain of Shh a of an on this we Shh in this of Shh, which we these and these or Shh, were into and Shh in both the and the of the the of the Shh from Shh into ShhNp with this of ShhNp were in the of cells with these which the sufficient ShhNp in the to by of Shh the crystal structure of that amino-terminal amino of Shh are in that s-ShhNp, we identify a amino required for this we a of amino-terminal to these Shh have on s-ShhNp formation. these we our to the palmitoylation of Shh, which plays an important role in formation and its activity Furthermore, the ability of ShhNp to is in amino-terminal that amino the for our R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). amino we or amino cells were with Shh, or and Shh in both and Shh, and were in the as both and cholesterol modified Shh were and were also in the of the in to Shh. The also as its form to its cholesterol modified with this of in the of Although from cells Shh the cells with the from cells with the Shh amino-terminal activity We on the from cells with The ability of to form to wt, with in its ability to form s-ShhNp. The ability of to form appeared to These results are with the highly conserved amino-terminal of Shh an important role in Shh Here, we show that of ShhNp are from a of for the and of s-ShhNp. Although Shh in as a multimeric s-ShhNp, or as a less we show that the of Shh activity with s-ShhNp. The s-ShhNp is as high concentrations of or have on its multimeric that types of interactions are required to this with this we show that s-ShhNp formation palmitoylation of Furthermore, we now provide based on interactions observed in the crystal structure of human R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), that s-ShhNp formation the of its highly conserved amino-terminal Thus, we for the first evidence for the existence of s-ShhNp based on the structural of human our results show that both protein-protein and protein-lipid interactions are required for s-ShhNp formation. analyses show that of ShhNp are required for s-ShhNp formation, amino amino and the palmitate site These first of Shh were to in Shh-Shh interactions based on the crystal structure of R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This structure that and hydrophobic with a second Shh and of or or of amino ShhNp less to form s-ShhNp. Although the ability of all of these Shh to form s-ShhNp is in to the of ShhNp to form s-ShhNp. into and to also form an Shh that form s-ShhNp into its These results are with of amino a on s-ShhNp formation of or as that and are both in which a of amino is is less multimeric we that amino also play an important structural role in s-ShhNp formation. of Shh amino that may to s-ShhNp formation is the domain Y. Development. 2002; 129: PubMed Google Scholar). of amino this highly conserved the high of with The biological activity of these Shh is in is to in that interactions are important for specific of Shh has been previously shown that is to form that are through the interactions of with a D.M. R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we that the also play a role in Shh-Shh through with a Although we have evidence for of that the formation of s-ShhNp, that Shh-Shh interactions and that the site of it remains that both of a palmitoylation of Shh it to Shh-Shh interactions to a of this the multimeric state of the Shh with of However, analyses of the palmitoylation state of Shh with state of s-ShhNp of of in a previously suggested that palmitoylation of ShhNp is important for s-ShhNp formation, a that has for a of (23.Chen M.H. Li Y.J. Kawakami T. Xu S.M. Chuang P.T. Genes Dev. 2004; 18: 641-659Crossref PubMed Scopus (293) Google Scholar). the of ShhNp to ShhNp shown in the Shh, with the of ShhNp the the multimeric to that palmitoylation for s-ShhNp formation. However, of the observed with Shh, it that the a on and that this in based on the of a Shh in which the palmitate into a a that to also function of Shh to palmitoylation (22.Taylor F.R. Wen D. Garber E.A. Carmillo A.N. Baker D.P. Arduini R.M. Williams K.P. Weinreb P.H. Rayhorn P. Hronowski X. Whitty A. Day E.S. Boriack-Sjodin A. Shapiro R.I. Galdes A. Pepinsky R.B. Biochemistry. 2001; 40: 4359-4371Crossref PubMed Scopus (132) Google Scholar). the activity of recombinant or from are the potency of that of recombinant is the activity of the to the to structural role that the amino terminus of Shh the in that the the structure in its ability to form multimers. the that palmitoylation of ShhNp is important for s-ShhNp formation, by a J. T. Lee J.D. Development. 2004; PubMed Scopus (68) Google Scholar). this second the of ShhNp in the to as a The results in this were of the of the which a high ShhNp in results show that palmitoylation of ShhNp is required for s-ShhNp formation. We results showing that the of ShhNp in is in a multimeric with a form of ShhNp as a in which the of to is is we also a for its ability to form and that this also to form s-ShhNp, as a This have on the structure and activity of Shh (22.Taylor F.R. Wen D. Garber E.A. Carmillo A.N. Baker D.P. Arduini R.M. Williams K.P. Weinreb P.H. Rayhorn P. Hronowski X. Whitty A. Day E.S. Boriack-Sjodin A. Shapiro R.I. Galdes A. Pepinsky R.B. Biochemistry. 2001; 40: 4359-4371Crossref PubMed Scopus (132) Google Scholar). we that palmitoylation of ShhNp is important for s-ShhNp formation. The our and that of J. T. Lee J.D. Development. 2004; PubMed Scopus (68) Google Scholar) are that it is to the in our However, the analyses is the of cells used to s-ShhNp. it remains that types have the ability to form s-ShhNp in a of is into the Shh that it is in a to Shh, the with in signaling (23.Chen M.H. Li Y.J. Kawakami T. Xu S.M. Chuang P.T. Genes Dev. 2004; 18: 641-659Crossref PubMed Scopus (293) Google Scholar). with that of the that formation of s-ShhNp palmitoylation of these results that the function of s-ShhNp is to act directly in Shh is with this as we show that the of ShhNp activity in is multimeric and that required for the signaling form of Shh. Furthermore, we are to the multimeric state of s-ShhNp from its biological the potency of is it is capable of s-ShhNp. we are also to forms of as that are as as s-ShhNp are as This potency and state of s-ShhNp that the of Shh is to its Thus, our results are with the function of s-ShhNp to its in a or a that the function of s-ShhNp is to ShhNp in a form capable of through the extracellular on the crystal structure of recombinant human R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), we amino-terminal of Shh important for s-ShhNp formation. Furthermore, we also that palmitoylation of ShhNp important for its These conclusions inconsistent with of that that and that recombinant X. Goetz J.A. Suber L.M. C.M. Robbins D.J. Nature. 2001; PubMed Scopus Google Scholar, J. T. Lee J.D. Development. 2004; PubMed Scopus (68) Google Scholar). We that the high protein concentrations required for protein may form the physiological for lipid of s-ShhNp. in Shh concentrations are palmitoylation of ShhNp required to the protein-protein interactions the Shh the structure of human previously that of that of which to the palmitate that R.B. Rayhorn P. Day E.S. A. Williams K.P. Galdes A. Taylor F.R. Boriack-Sjodin P.A. Garber E.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Although has less activity ShhN, it the high protein concentrations in the the ability of the palmitate to the Shh interactions in s-ShhNp. We have of this structure to provide the first structural evidence that the existence of a multimeric form of s-ShhNp. We are to members of the Robbins and to of for the of this We are also to and Zeng for to the development of the described