SUPPLEMETARY FIGURE LEGEDS Supplementary Fig. 1. Flow cytometric analysis of wildtype, mutant and chimeric protein surface expression. Cells transduced with the individual constructs indicated were stained for CD69 or KRp1A ectodomains, or Flag in the case of S1P-receptor constructs. Histogram overlays group constructs in accord with their appearance in Western blots in the designated figure. Dot plots show flow cytometric analysis of Flag-S1P 1 and CD69 on the indicated WEHI231 cell lines that correspond to the cells used for the binding studies in Figure 10. Supplementary Fig. 2. Point mutations of all residues in the HEGSI motif of CD69 to alanine. on-specific bands are indicated with an asterisk. Supplementary Fig. 3. A. Flow cytometric analysis of S1P 1 mutants with point mutations in helical segments co-transduced with CD69 and hkrp1a in WEHI231 cells. The cells are costained for CD69 and KRp1A ectodomains and the Flag-S1P receptors and mutants indicated. B. Co-IP of the mutants indicated. o mutant (P1 through P8) exhibited a more than 1.5 fold difference in HA/Flag signal by densitometry. The result is representative of two experiments. on-specific bands are indicated with an asterisk. Supplementary Fig. 4. Test of electrostatics of S1P 1 and S1P 3. Structure was generated with modeller using the most homologous GPCR structure (β1-adrenergic receptor, pdb ID 2VT4). The region of the GPCR represented by helix 4 is highlighted by a black oval. Other helices on this surface are annotated. 1
Supplementary Figure 1 2
Supplementary Figure 2 ame Ectodomain Stalk Transmembrane Intracellular S1P X 20% Input WB: Flag CD69 C H32A H33A E34A G35A S36A I37A KRp1A C C C C C C C Ala Ala Ala 1 3 1 3 1 3 1 3 1 3 1 3 1 3 1 Ala Ala Ala CD69 or KRp1A Construct 20% Input WB: HA 20% Input WB: Actin IP: Flag WB: Flag IP: Flag WB: HA * * 3
Supplementary Figure 3 A P1 P2 P3 10 12 5 24 12.6 16.7 8.19 19.9 10 4 P1 P2 P3 10 3.57 5 70.9 6.19 71.3 3.33 65.8 10 4 10 3 10 3 10 2 0 13.7 50.3 21.8 48.8 16.4 55.5 10 2 0 2.05 23.5 3.55 19 2.9 28 CD69 ectodomain 10 5 11.9 21.1 10 4 10 3 10 2 0 10 5 25.3 6.45 10 4 10 3 10 2 0 21.4 32.6 P4 P5 P6 45.6 35.7 12.1 12.3 26.2 P7 P8 9.74 19.2 19.5 49.4 51.5 Flag-S1P receptor 9.76 18 18.3 54 3.4 28.4 6.81 61.4 KRp1A ectodomain 10 5 5.13 71.3 10 4 10 3 10 2 0 10 5 24.2 52.8 10 4 10 3 10 2 0 3.58 10.8 P4 P5 P6 20 12.3 6.35 68.2 3.6 9.96 65.8 4.88 21.9 19.3 6.81 69.4 3.73 20.1 Edg1 P7 P8 Edg1 Flag-S1P receptor 2.01 66 1.22 30.7 B ame S1P1 P1 P2 S1P3 S1P1 P3 P4 P5 P6 P7 P8 S1P3 69 K 69 K 69 K 69 69 K 69 K 69 K 69 K 69 K 69 K 69 K 69 20% Input WB: Flag * 20% Input WB: HA * 20% Input WB: Actin IP: Flag WB: Flag IP: Flag WB: HA * * 4
Supplementary Figure 4 S1P 1 S1P 3 Helix 4 Helix 5 Helix 4 Helix 5 Helix 3 Helix 3 5
SUPPLEMETARY EXPERIMETAL PROCEDURES Protein sequence of constructs used in this paper: S1P receptor constructs S1P1 (CBI Reference Sequence: P_031927.2) sphingosine-1-phosphate receptor 1 [Mus musculus] MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS S1P3 (CBI Reference Sequence: P_034231.1) sphingosine-1-phosphate receptor 3 [Mus musculus] MATTHAQGHQPVLGDTLREHYDYVGKLAGRLRDPPEGGTLITTILFLVTCSFIVLELMVLIAIWKK FHRMYFFIGLALCDLLAGIAYKVILMSGRKTFSLSPTVWFLREGSMFVALGASTCSLLAIAIERHLT MIKMRPYDAKKHRVFLLIGMCWLIAFSLGALPILGWCLEFPDCSTILPLYSKKYIAFLISIFTAILV TIVILYARIYCLVKSSSRRVAHSERSMALLRTVVIVVSVFIACWSPLFILFLIDVACRAKECSILFKS QWFIMLAVLSAMPVIYTLASKEMRRAFFRLVCGCLVKGKGTQASPMQPALDPSRSKSSSSSSHSPK VKEDLPRVATSSCIIDKRSFQGVLCK S5A (1) MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF ARAKADAAHPQKDDGDPETIMSSGVSSS Δ12 (2) MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPE R120A (3) MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLAEGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS Y-sulfo (4) MVSTSIPEVKALRSSVSDFGFDIIVRHFFTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLAEGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS 6
30D (5) MVSTSIPEVKALRSSVSDYGYDIIVRHYDYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS S1P1+3 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIK ITTILFLVTCSFIVLELMVLIAIWKK FHRMYFFIGLALCDLLAGIAYKVILMSGRKTFSLSPTVWFLREGSMFVALGASTCSLLAIAIERHLT MIKMRPYDAKKHRVFLLIGMCWLIAFSLGALPILGWCLEFPDCSTILPLYSKKYIAFLISIFTAILV TIVILYARIYCLVKSSSRRVAHSERSMALLRTVVIVVSVFIACWSPLFILFLIDVACRAKECSILFKS QWFIMLAVLSAMPVIYTLASKEMRRAFFRLVCGCLVKGKGTQASPMQPALDPSRSKSSSSSSHSPK VKEDLPRVATSSCIIDKRSFQGVLCK S1P3+1 MATTHAQGHQPVLGDTLREHYDYVGKLAGRLRDPPEGGTL LTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS S1P31313 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIK ITTILFLVTCSFIVLELMVLIAIWKK FHRMYFFIGLALCDLLAGIAYKVILMSGRKTFSLSPTVWFLREGSMFVALGASTCSLLAIAIERHLT MIKMRPYDAKKHRVFLLIGMCWLIAFSLGALPILGWCLEFPDCSTILPLYSKKYIAFLISIFTAILV TIVILYARIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVVIVVSVFIACWSPLFILFLIDVACRAK ECSILFKSQWFIMLAVLSAMPVIYTLASKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEFSRSKS DSSHPQKDDGDPETIMSSGVSSS R142A MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI EAYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS R142 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI EYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS 7
A MATTHAQGHQPVLGDTLREHYDYVGKLAGRLRDPPEGGTLITTILFLVTCSFIVLELMVLIAIWKK FHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS B MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHHRMYFFIGLALCDLLAGIAYKVILMSGRKTFSLSPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS C MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTAPTVWFLREGSMFVALGASTCSLLAIAIERHLT MIKMRPYDASSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS D MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSKKHRVFLLIGMCWLIAFSLGALPILGWCLEFPDCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS E MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTILPLYSKKYIAFLISIFTAILVTI VILYARIYCLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS F MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVKSSSRRVAHSERSMALLRTVVIVVSVFIACWSPLFILFLIDVACRAKECSILYKAEY FLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS G MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILFKSQWFIMLAVLSAMPVIYTLASKEMRRAFFRLVCGCLVKGKGTQASPMQPALDPSRSKSSSS SSHSPKVKEDLPRVATSSCIIDKRSFQGVLCK 8
D1 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSKKHRVFLLISACWVISLILGGLPIM GWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS D2 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLIGMCWLIAFSLGALPILGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS D3 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPIMGWCLEFPDCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS S1P31D MATTHAQGHQPVLGDTLREHYDYVGKLAGRLRDPPEGGTLITTILFLVTCSFIVLELMVLIAIWKK FHRMYFFIGLALCDLLAGIAYKVILMSGRKTFSLSPTVWFLREGSMFVALGASTCSLLAIAIERHLT MIKMRPYDAKKHRVFLLISACWVISLILGGLPIMGWCLEFPDCSTILPLYSKKYIAFLISIFTAILV TIVILYARIYCLVKSSSRRVAHSERSMALLRTVVIVVSVFIACWSPLFILFLIDVACRAKECSILFKS QWFIMLAVLSAMPVIYTLASKEMRRAFFRLVCGCLVKGKGTQASPMQPALDPSRSKSSSSSSHSPK VKEDLPRVATSSCIIDKRSFQGVLCK P1 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLIGACWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS P2 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISMCWVISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS 9
P3 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWLISLILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS P4 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVIALILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS P5 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISFILGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS P6 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLSLGGLPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS P7 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGALPIMGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS P8 MVSTSIPEVKALRSSVSDYGYDIIVRHYYTGKLIGAEKDHGIKLTSVVFILICCFIILEIFVLLTI WKTKKFHRPMYYFIGLALSDLLAGVAYTALLLSGATTYKLTPAQWFLREGSMFVALSASVFSLLAIAI ERYITMLKMKLHGSSSRSFLLISACWVISLILGGLPILGWCISSLSSCSTVLPLYHKHYILFCTTVF TLLLLSIVILYCRIYSLVRTRSRRLTFRKISKASRSSEKSLALLKTVIIVLSVFIACWAPLFILLLLDV GCKAKTCDILYKAEYFLVLAVLSGTPIIYTLTKEMRRAFIRIVSCCKCPGDSAGKFKRPIIPGMEF SRSKSDSSHPQKDDGDPETIMSSGVSSS C-type lectin chimeric constructs CD69 (CBI Reference Sequence: P_001028294.1) MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 10
hkrp1a (CBI Reference Sequence: P_002249.1) MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSVSVTSLIQKSSIEKCSVD IQQSRKTTERPGLLCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWI GLFSLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS 69IT-KE MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVQKSSIEKCSVDIQQ SRKTTERPGLLCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWIGL FSLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS KIT-69E MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSPWHQFALKLSCAGIILLVLVVTGLSVSVTSLIGKYCPGLYEK LESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQ TWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 69I-KTE MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQLSCAGIILLVLVVTGLSVSVTSLIQKSSIEKCSVDIQQS RKTTERPGLLCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWIGLF SLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS KI-69TE MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSPWHQFALKVSIPWAVLIVVLITSLIIALIALVGKYCPGLYE KLESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEA QTWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 66 MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQLSCAGIILLVLVVTGLSVSVTSLIGKYCPGLYEKLESS DHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWKW AGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 6 MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSPWHQFALKVSIPWAVLIVVLITSLIIALIALVQKSSIEKCSV DIQQSRKTTERPGLLCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFW IGLFSLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS 66-Δ31 MDSECSITESSSHLERGQKDHGTSIHFEKPWHQFALKLSCAGIILLVLVVTGLSVSVTSLIGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 66-stalk MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQLSCAGIILLVLVVTGLSVSVTSLISSIEKCSVDIQQSR KTTERPGLLCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQ TWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 66-DS MDSECSITESSSHLERGQKDHGTSIHFEKPWHQFALKLSCAGIILLVLVVTGLSVSVTSLISSIEKCSVDIQQSR KTTERPGLLCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEA QTWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 11
6-Δ31 MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSHHEGSIQVSIPWAVLIVVLITSLIIALIALVQKSSIEKCSVD IQQSRKTTERPGLLCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWI GLFSLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS 6-stalk MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSHHEGSIQVSIPWAVLIVVLITSLIIALIALVQKGKYCPGLY EKLESSDHHVATCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWIGLF SLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS 6-DS MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSPWHQFALKVSIPWAVLIVVLITSLIIALIALVQKGKYCPGL YEKLESSDHHVATCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWIGL FSLSEKWKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS 69-KS1 MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVQKSSIEKCSVDIQQ SKLESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEA QTWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 69-KS2 MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYERKT TERPGLLCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTW KWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 69-KS12 MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVQKSSIEKCSVDIQQ SRKTTERPGLLCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKE AQTWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 69-KS1 MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVQKSSIEKCPGLYEK LESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQ TWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 69-KS1C MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCSVDIQQSKL ESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQT WKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 69IS-KE MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCPIYWQQLREKCLLFSHTVPWSLADCSTKESSLLLIRDKDELIHTQLIRDKAILFWIGLFSLSEK WKWIGSFLSDLEIRGDAKESCISISQTSVYSEYCSTEIRWICQKELTPVRKVYPDS KD-69TE MDQQAIYAELLPTDSGPESSSPSSLPRDVCQGSHHEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEK LESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQ TWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 12
PWHQFA-Insert MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSIPWHQFAQVSIPWAVLIVVLITSLIIALIALVGKYCPGL YEKLESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKE AQTWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR HEGSI-delete MDSECSITESSSHLERGQKDHGTSIHFEKH- QVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLESSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCS EDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWKWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFH WVCSKPSR HESGI-replace MDSECSITESSSHLERGQKDHGTSIHFEKHPWHQFAQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLE SSDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTW KWAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR H32A MDSECSITESSSHLERGQKDHGTSIHFEKAHEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR H33A MDSECSITESSSHLERGQKDHGTSIHFEKHAEGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR E34A MDSECSITESSSHLERGQKDHGTSIHFEKHHAGSIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR G35A MDSECSITESSSHLERGQKDHGTSIHFEKHHEASIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR S36A MDSECSITESSSHLERGQKDHGTSIHFEKHHEGAIQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR I37A MDSECSITESSSHLERGQKDHGTSIHFEKHHEGSAQVSIPWAVLIVVLITSLIIALIALVGKYCPGLYEKLES SDHHVATCKEWISYKRTCYFFSTTTKSWALAQRSCSEDAATLAVIDSEKDMTFLKRYSGELEHWIGLKEAQTWK WAGKEFSWFLTGSGRCVSVHKVTAVDCEAFHWVCSKPSR 13
SUPPLEMETARY REFERECES 1. Oo, M. L., Thangada, S., Wu, M. T., Liu, C. H., Macdonald, T. L., Lynch, K. R., Lin, C. Y., and Hla, T. (2007) J Biol Chem 282, 9082-9089 2. Watterson, K. R., Johnston, E., Chalmers, C., Pronin, A., Cook, S. J., Benovic, J. L., and Palmer, T. M. (2002) J Biol Chem 277, 5767-5777 3. Parrill, A. L., Wang, D., Bautista, D. L., Van Brocklyn, J. R., Lorincz, Z., Fischer, D. J., Baker, D. L., Liliom, K., Spiegel, S., and Tigyi, G. (2000) J Biol Chem 275, 39379-39384 4. Fieger, C. B., Huang, M. C., Van Brocklyn, J. R., and Goetzl, E. J. (2005) FASEB J 19, 1926-1928 5. Kohno, T., Wada, A., and Igarashi, Y. (2002) FASEB J 16, 983-992 14