Fig 1.
Alignment of the amino acid sequence of zfGLP-1 with sequences of hGLP-1, zebrafish glucagon, human glucagon, exendin-4, exendin(9–39), zfGLP-2 and zebrafish PACAP-38.
Identical amino acids are shown in red. Numbering of hGLP-1 starts at 1 with the amino terminal histidine, corresponding to the biologically active hGLP-1(7–37) and hGLP-1(7–36)amide, to be consistent with histidine 1 in zfGLP-1 (see Materials and Methods).
Fig 2.
Snake diagram of the zfGPCR (dual zfGLP-1R/GCGR).
Amino acids are numbered in the N-terminal extracellular domain (NECD) according to the numbering in the crystal structure of the NECD of hGLP-1R in complex with hGLP-1 (PDB entry 3IOL) [36]. Numbering of amino acids in the 7TM domain and the C-terminal cytoplasmic domain is according to the numbering in the 7TM crystal structure of hGCGR (PDB entry 4L6R) [30]. The following amino acids are numbered: (i) position 32 (glutamine) at the beginning of the predicted amphiphilic helix in zfGPCR corresponding to Leu32 at the beginning of the amphiphilic helix in hGLP-1R and Met32 at the beginning of the amphiphilic helix of hGCGR (see Fig 3 and S1 Fig); (ii) the eight cysteine residues forming the four disulfide bonds as indicated by yellow lines: (iii) glutamic acid in the cytoplasmic domain at position 406 at the beginning of helix 8 identified in the 7TM crystal structure of hGCGR (PDB entry 4L6R) [30]. Residues with similar physicochemical properties are colored with identical colors according to the residue-based diagram editor RbDe for GPCRs [44].
Fig 3.
Multiple sequence alignment of the N-terminal extracellular domains (NECD) of zfGPCR, hGLP-1R and hGCGR.
Numbering of amino acids is according to the numbering in the crystal structure of the NECD of hGLP-1R in complex with hGLP-1 (PDB entry 3IOL) [36]. The α-helical region is shaded grey, loops are colored pink, and the stalk region is shaded green. Note the absence of loop 3 in zfGPCR. The interhelical salt bridge in the α-helical stalk region in hGCGR identified in the 7TM crystal structure of hGCGR (PDB entry 4L6R) [30] is indicated at the end of the α- helical stalk region by a line. Residues with stabilizing functions are colored blue. Residues forming the exendin(9–39) hydrophobic binding pocket in the hGLP-1R NECD [35] (PDB entry 3C59) are colored in yellow. Residues which are both part of the binding pocket and have stabilizing functions are colored with blue and yellow hatching. Residues that are part of the hydrogen bond network in hGLP-1R coordinated by Asp67 [36] (PDB entry 3IOL) [35] (PDB entry 3C59) are marked with a hash sign. Cysteines are colored orange and paired cysteines are denoted by similar outlines (Fig 2). Conserved tryptophan residues are highlighted with an asterisk below the alignment.
Fig 4.
Amino acids in the hGLP-1R NECD hydrophobic cluster are primarily aromatic (blue color) while the corresponding residues in zfGPCR (green and magenta colors) are aliphatic (Fig 3), but likely do not affect the direct interaction of zfGPCR with exendin(9–39) (gold) or hGLP-1 (not shown) peptides.
The conserved Tyr101 is not shown on the diagram for emphasis on differences. Cyan color represents the structure of the hGLP-1R NECD as bound to exendin(9–39) [35] (and PDB entry 3C59), or in light blue as bound to hGLP-1 [36] (and PDB 3IOL), and green the predicted zfGPCR structure based on the structure of the hGLP-1R NECD in complex with exendin(9–39) [35] (PDB entry 3C59). Backbone of residue Gly120 in zfGPCR (instead of Trp120 in hGLP-1R) is shown in magenta to highlight the lack of side chain. Note the absence of loop 3 in zfGPCR. Gln112 in hGLP-1R is a histidine in zfGPCR (Fig 3) maintaining similar hydrophobicity characteristics.
Fig 5.
Substitution of Arg121 with lysine in zfGPCR and the absence of loop 3 should not affect the hydrogen bonding and the salt bridge network found in the crystal structures of the NECD of hGLP-1R in complex with exendin(9–39) or GLP-1 The hydrogen bond interaction between Arg121 in hGLP-1R with the main chain oxygen of position 27 in hGLP-1 (3.0Å) and exendin(9–39) (2.8Å, not shown) can be maintained by the conserved substitution of arginine with lysine in zfGPCR (Fig 3).
Light blue ribbon diagram represents the structure of the hGLP-1R NECD bound to GLP-1 [36] (PDB entry 3IOL) and green the predicted structure of zfGPCR. Lys121 in zfGPCR was modeled from the structure of the hGLP-1R NECD in complex with hGLP-1 [36] (PDB entry 3IOL) by removing the missing residues and closing the gap with PyMOL’s sculpting module.
Fig 6.
Multiple sequence alignment of TM helices and cytoplasmic helix 8 in zfGPCR, hGLP-1R and hGCGR.
The most conserved residues in each TM are labeled using the modified Ballesteros-Weinstein generic numbering system [42, 43] (see Materials and Methods) and numbered according to the 7TM crystal structure of hGCGR [30] (PDB entry 4L6R). Conserved interactions between residues in different TM helices are highlighted in similar colors. The hydrogen bond network coordinated by Glu2453.50 (see Fig 7) is shown in dark blue. Residues outside of the TM domains are shaded light grey to highlight the conserved Arg residue that interacts with Glu406 (superscript numbers) in cytoplasmic helix 8 and are labeled and shaded dark grey.
Fig 7.
The TM2-TM3-TM6-TM7 hydrogen bond network coordinated by Glu2453.50 in TM3 with residues in TM6, TM2 and TM7 may contribute to the stability of the protein and thereby affect cell surface expression of hGCGR, zfGPCR and hGLP-1R (the contacts seen are those on the hGCGR, PDB entry 4L6R).
In hGLP-1R, the mutation to alanine of Glu2453.50 in TM3 (Panel B), of His1772.50 in TM2 (Panel C) and of Tyr4007.57 in TM7 (Panel D) significantly reduced cell surface expression of GLP-1R [42]. Binding of 125I-exendin(9–39) to hGLP-1R could not be measured in these hGLP-1R mutants [42]. Alanine in these positions (Panels B, C, D) would not maintain the hydrogen bond network between all four transmembrane helices seen in the 7TM crystal structure of hGCGR.
Fig 8.
The predicted TM4-TM3-TM6 helical bundle would be more compact in zfGCGR than in hGGCR while in hGLP-1R is likely the most expanded.
Cys2403.45 in TM3 of hGCGR [30] (PDB entry 4L6R; brown) forms side chain-to-backbone interaction with Gly2714.49 in TM4. Substitution of this cysteine with a tyrosine in hGLP-1R (magenta) should shift TM4 away from TM3 and TM6. TM3-TM6 interactions (box) of hGCGR between Tyr2393.44 in TM3 and main chain atoms of Gly3596.50 and Leu3586.49 in TM6 will be maintained in hGLP-1R, which has the same tyrosine, but not in zfGPCR with an asparagine (green) at this position. The shorter asparagine side chain should bring TM6 closer to TM3 in order to maintain these interactions.
Table 1.
Summary of the sequence and structural mapping of the 7TM domains in zfGPCR and hGLP-1R onto hGCGR based on the 7TM structure of hGCGR ([30].
Table 2.
Summary of the sequence and structural mapping of the NECD of zfGPCR onto the NECDs of GLP-1R and hGCGR based on their crystal structures [32, 35, 36].
Table 3.
ZfGPCR does not significantly discriminate between zfGLP-1, hGLP-1, zebrafish glucagon and human glucagon, as determined from competitive binding experiments shown in Figs 9 and 10.
Fig 9.
Zebrafish glucagon displaces the binding of radioiodinated 125I-hGLP-1(7–36)amide to the recombinant zfGPCR in a similar dose-dependent manner as zfGLP-1, hGLP-1, exendin-4 and exendin(9–39).
Displacement curves represent average measurements obtained from three separate rounds of transfections for the displacement with hGLP-1 (n = 3), two for exendin-4, exendin(9–39) and zebrafish glucagon (n = 2) and one for zfGLP-1, zfGLP-2 and zebrafish PACAP-38 (n = 1). Data points for each concentration in each displacement curve obtained in a single round of transfection are an average of three independent measurements. Error bars are shown for data points in the displacement curves obtained in two or more rounds of transfections (see Materials and Methods).
Fig 10.
Higher concentrations of zfGLP-1, hGLP-1, zebrafish glucagon and human glucagon are required to displace 50% of the binding of 125I-exendin(9–39) to the recombinant zfGPCR relative to exendin-4 and exendin(9–39).
Compare to Fig 9. Displacement curves with zfGLP-1 represent average measurements obtained from four separate rounds of transfections (n = 4), for hGLP-1, zebrafish glucagon, human glucagon and exendin-4 from three (n = 3) and for exendin(9–39) from five (n = 5). Each data point in the displacement curve obtained in a single round of transfection is an average of three independent measurements.
Table 4.
Zebrafish glucagon and human glucagon stimulate intracellular cAMP mediated through the recombinant zfGPCR to a similar degree as zfGLP-1 and hGLP-1, as determined from the dose-response curves shown on Fig 11.
Fig 11.
ZfGLP-1 (n = 9), hGLP-1 (n = 5), zebrafish glucagon (n = 5), human glucagon (n = 5) and exendin-4 (n = 3) stimulate intracellular cAMP through the recombinant zfGPCR in a similar dose-dependent manner.
Exendin(9–39) (n = 4) and zebrafish PACAP-38 (n = 2) have no effect, and zfGLP-2 (n = 6) has stimulatory effects only at much higher concentrations than the other tested peptides that stimulated cAMP. (n) represents number of separate rounds of transfections. Each data point in the dose-response curve obtained in a single transfection is an average of three separate measurements (see Materials and Methods). To highlight differences between stimulatory effects of zfGLP-1, zebrafish glucagon, human GLP-1, human glucagon and exendin-4 from the stimulatory effect of zfGLP-2 error bars are shown only for zfGLP-1, zfGLP-2, exendin(9–39) and zfPACAP-38.