A confined variable region confers ligand specificity on fibroblast growth factor receptors: implications for the origin of the immunoglobulin fold.

Binding of cellular growth factors to their receptors constitutes a highly specific interaction and the basis for cell and tissue-type specific growth and differentiation. A unique feature of fibroblast growth factor (FGF) receptors is the multitude of structural variants and an unprecedented degree of cross-reactivity between receptors and their various ligands. To examine receptor-ligand specificity within these families of growth factors and receptors, we used genetic engineering to substitute discrete regions between Bek/FGFR2 and the closely related keratinocyte growth factor receptor (KGFR). We demonstrate that a confined, 50 amino acid, variable region within the third immunoglobulin-like domain of Bek and KGFR exclusively determines their ligand binding specificities. Replacing the variable region of Bek/FGFR2 with the corresponding sequence of KGFR resulted in a chimeric receptor which bound KGF and had lost the capacity to bind basic FGF. We present evidence that the two variable sequences are encoded by two distinct exons that map close together in the mouse genome and follow a constant exon, suggesting that the two receptors were derived from a common gene by mutually exclusive alternative mRNA splicing. These results identify the C-terminal half of the third immunoglobulin-like domain of FGF receptors as a major determinant for ligand binding and present a novel genetic mechanism for altering receptor-ligand specificity and generating receptor diversity.     

A novel form of fibroblast growth factor receptor 2. Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity.

The fibroblast growth factor receptor 2 (FGFR2) gene is expressed as alternatively spliced mRNAs that encode bacterially expressed kinase, the keratinocyte growth factor receptor, or K-sam. We have now isolated a novel FGFR2 cDNA that is identical with the previously cloned human bacterially expressed kinase, except in the third immunoglobulin-like domain. The ligand binding properties of FGFR2 were studied by expressing the protein in rat L6 muscle myoblasts. Unlike human bacterially expressed kinase which binds acidic and basic FGF with similar affinities, FGFR2 bound acidic FGF with approximately 1000-fold higher affinity than basic FGF. These results indicate that alternative splicing of the FGFR2 gene in the region encoding the carboxyl-terminal half of the third immunoglobulin domain determines the ligand specificity of this group of receptors.     

Identification of residues important both for primary receptor binding and specificity in fibroblast growth factor-7

Fibroblast growth factors (FGFs) mediate a multitude of physiological and pathological processes by activating a family of tyrosine kinase receptors (FGFRs). Each FGFR binds to a unique subset of FGFs and ligand binding specificity is essential in regulating FGF activity. FGF-7 recognizes one FGFR isoform known as the FGFR2 IIIb isoform or keratinocyte growth factor receptor (KGFR), whereas FGF-2 binds well to FGFR1, FGFR2, and FGFR4 but interacts poorly with KGFR. Previously, mutations in FGF-2 identified a set of residues that are important for high affinity receptor binding, known as the primary receptor-binding site. FGF-7 contains this primary site as well as a region that restricts interaction with FGFR1. The sequences that confer on FGF-7 its specific binding to KGFR have not been identified. By utilizing domain swapping and site-directed mutagenesis we have found that the loop connecting the beta4-beta5 strands of FGF-7 contributes to high affinity receptor binding and is critical for KGFR recognition. Replacement of this loop with the homologous loop from FGF-2 dramatically reduced both the affinity of FGF-7 for KGFR and its biological potency but did not result in the ability to bind FGFR1. Point mutations in residues comprising this loop of FGF-7 reduced both binding affinity and biological potency. The reciprocal loop replacement mutant (FGF2-L4/7) retained FGF-2 like affinity for FGFR1 and for KGFR. Our results show that topologically similar regions in these two FGFs have different roles in regulating receptor binding specificity and suggest that specificity may require the concerted action of distinct regions of an FGF.     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.     

The second immunoglobulin-like domain of the VEGF tyrosine kinase receptor Flt-1 determines ligand binding and may initiate a signal transduction cascade.

Vascular endothelial growth factor (VEGF) is an angiogenic inducer that mediates its effects through two high affinity receptor tyrosine kinases, Flt-1 and KDR. Flt-1 is required for endothelial cell morphogenesis whereas KDR is involved primarily in mitogenesis. Flt-1 has an alternative ligand, placenta growth factor (PlGF). Both Flt-1 and KDR have seven immunoglobulin (Ig)-like domains in the extracellular domain. The significance and function of these domains for ligand binding and receptor activation are unknown. Here we show that deletion of the second domain of Flt-1 completely abolishes the binding of VEGF. Introduction of the second domain of KDR into an Flt-1 mutant lacking the homologous domain restored VEGF binding. However, the ligand specificity was characteristic of the KDR receptor. We then created chimeric receptors where the first three or just the second Ig-like domains of Flt-1 replaced the corresponding domains in Flt-4, a receptor that does not bind VEGF, and analyzed their ability to bind VEGF. Both swaps conferred upon Flt-4 the ability to bind VEGF with an affinity nearly identical to that of wild-type Flt-1. Furthermore, transfected cells expressing these chimeric Flt-4 receptors exhibited increased DNA synthesis in response to VEGF or PlGF. These results demonstrate that a single Ig-like domain is the major determinant for VEGF-PlGF interaction and that binding to this domain may initiate a signal transduction cascade.     

Interspecies molecular chimeras of kit help define the binding site of the stem cell factor.

The extracellular portion of the kit-encoded receptor for the stem cell factor (SCF) comprises five immunoglobulin (Ig)-like domains. To localize the ligand recognition site, we exploited the lack of binding of human SCF to the murine receptor by using human-mouse hybrids of Kit and species-specific monoclonal antibodies (MAbs) that inhibit ligand binding. Replacement of the three N-terminal Ig-like domains of the murine Kit with the corresponding portion of the human receptor conferred upon the chimeric receptor high-affinity binding of the human ligand as well as of human-specific ligand-inhibitory MAbs. By constructing five chimeric murine Kit proteins which individually contain each of these three human Ig-like units or pairs of them, we found that the second human domain confers upon the mouse Kit high-affinity binding of the human ligand and also binding of species-specific SCF-competitive MAbs. Nevertheless, the flanking Ig-like domains also affect high-affinity recognition of SCF. Moreover, it appears that the determinants that define ligand specificity of the murine and the human receptors do not structurally coincide. This observation allowed us to identify a chimeric receptor that displayed a dual specificity; namely, it bound with high affinity either the human or the murine SCF molecules and reacted with mouse- as well as human-specific ligand-inhibitory MAbs. Conversely, another chimera, which included all of the five Ig-like domains, bound neither ligand. In conclusion, interdomain packing involving the second Ig-like domain of human Kit and noncontiguous structural motifs of the receptor are involved in SCF recognition.     

Characterization of a second ligand binding site of the insulin receptor

Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the alpha subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K(d) of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site.     

Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity

To elucidate the structural determinants governing specificity in fibroblast growth factor (FGF) signaling, we have determined the crystal structures of FGF1 and FGF2 complexed with the ligand binding domains (immunoglobulin-like domains 2 [D2] and 3 [D3]) of FGF receptor 1 (FGFR1) and FGFR2, respectively. Highly conserved FGF-D2 and FGF-linker (between D2-D3) interfaces define a general binding site for all FGF-FGFR complexes. Specificity is achieved through interactions between the N-terminal and central regions of FGFs and two loop regions in D3 that are subject to alternative splicing. These structures provide a molecular basis for FGF1 as a universal FGFR ligand and for modulation of FGF-FGFR specificity through primary sequence variations and alternative splicing.     

Identification and characterization of two distinct ligand binding regions of cubilin

Using polymerase chain reaction-amplified fragments of cubilin, an endocytic receptor of molecular mass 460 kDa, we have identified two distinct ligand binding regions. Region 1 of molecular mass 71 kDa, which included the 113-residue N terminus along with the eight epidermal growth factor (EGF)-like repeats and CUB domains 1 and 2, and region 2 of molecular mass 37 kDa consisting of CUB domains 6-8 bound both intrinsic factor-cobalamin (vitamin B(12); Cbl) (IF-Cbl) and albumin. Within these two regions, the binding of both ligands was confined to a 110-115-residue stretch that encompassed either the 113-residue N terminus or CUB domain 7 and 8. Ca(2+) dependence of ligand binding or the ability of cubilin antiserum to inhibit ligand binding to the 113-residue N terminus was 60-65%. However, a combination of CUB domains 7 and 8 or 6-8 was needed to demonstrate significant Ca(2+) dependence or inhibition of ligand binding by cubilin antiserum. Antiserum to EGF inhibited albumin but not IF-Cbl binding to the N-terminal cubilin fragment that included the eight EGF-like repeats. While the presence of excess albumin had no effect on binding to IF-Cbl, IF-Cbl in excess was able to inhibit albumin binding to both regions of cubilin. Reductive alkylation of the 113-residue N terminus or CUB 6-8, CUB 7, or CUB 8 domain resulted in the abolishment of ligand binding. These results indicate that (a) cubilin contains two distinct regions that bind both IF-Cbl and albumin and that (b) binding of both IF-Cbl and albumin to each of these regions can be distinguished and is regulated by the nonassisted formation of local disulfide bonds.     

Structural requirements for ligand binding by a probable plant vacuolar sorting receptor

How sorting receptors recognize amino acid determinants on polypeptide ligands and respond to pH changes for ligand binding or release is unknown. The plant vacuolar sorting receptor BP-80 binds polypeptide ligands with a central Asn-Pro-Ile-Arg (NPIR) motif. tBP-80, a soluble form of the receptor lacking transmembrane and cytoplasmic sequences, binds the peptide SSSFADSNPIRPVTDRAASTYC as a monomer with a specificity indistinguishable from that of BP-80. tBP-80 contains an N-terminal region homologous to ReMembR-H2 (RMR) protein lumenal domains, a unique central region, and three C-terminal epidermal growth factor (EGF) repeats. By protease digestion of purified secreted tBP-80, and from ligand binding studies with a secreted protein lacking the EGF repeats, we defined three protease-resistant structural domains: an N-terminal/RMR homology domain connected to a central domain, which together determine the NPIR-specific ligand binding site, and a C-terminal EGF repeat domain that alters the conformation of the other two domains to enhance ligand binding. A fragment representing the central domain plus the C-terminal domain could bind ligand but was not specific for NPIR. These results indicate that two tBP-80 binding sites recognize two separate ligand determinants: a non-NPIR site defined by the central domain-EGF repeat domain structure and an NPIR-specific site contributed by the interaction of the N-terminal/RMR homology domain and the central domain.     

Binding of leukemia inhibitory factor (LIF) to mutants of its low affinity receptor, gp190, reveals a LIF binding site outside and interactions between the two cytokine binding domains.

The gp190 transmembrane protein, the low affinity receptor for the leukemia inhibitory factor (LIF), belongs to the hematopoietin family of receptors characterized by the cytokine binding domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proximal CBD (herein called D2) is separated from the membrane-distal one (called D1) by an immunoglobulin-like (Ig) domain and is followed by three fibronectin type III repeats. We used truncated gp190 mutants and a blocking anti-gp190 monoclonal antibody to study the role of these repeats in low affinity receptor function. Our results showed that the D1Ig region was involved in LIF binding, while D2 appeared to be crucial for the proper folding of D1, suggesting functionally important interactions between the two CBDs in the wild-type protein. In addition, a point mutation in the carboxyl terminus of the Ig region strongly impaired ligand binding. These findings suggest that at least two distinct sites, both located within the D1Ig region, are involved in LIF binding to gp190, and more generally, that ligand binding sites on these receptors may well be located outside the canonical CBDs.     

Solution structure of the ligand binding domain of the fibroblast growth factor receptor: role of heparin in the activation of the receptor

The three-dimensional solution structure of the ligand binding D2 domain of the fibroblast growth factor receptor (FGFR) is determined using multidimensional NMR techniques. The atomic root-mean-square distribution for the backbone atoms in the structured region is 0.64 A. Secondary structural elements in the D2 domain include 11 beta-strands arranged antiparallely into two layers of beta-sheets. The structure of the D2 domain is characterized by the presence of a short flexible helix that protrudes out of the layers of beta-sheets. Results of size exclusion chromatography and sedimentation velocity experiments show that the D2 domain exists in a monomeric state both in the presence and in the absence of bound sucrose octasulfate (SOS), a structural analogue of heparin. Comparison of the solution structure of the D2 domain with the crystal structure of the protein (D2 domain) in the FGF signaling complex reveals significant differences, suggesting that ligand (FGF) binding may induce significant conformational changes in the receptor. SOS binding sites in the D2 domain have been mapped on the basis of the 1H-15N chemical shift perturbation data. SOS binds to the positively charged residues located in beta-strand III and the flexible helix. Isothermal titration calorimetry data indicate that the ligand (hFGF-1) binds strongly (Kd approximately 10(-9) M) to the D2 domain even in the absence of SOS. Binding of SOS to either the D2 domain or hFGF-1 does not seem to be the driving force for the formation of the D2-hFGF-1 binary complex. The function of SOS binding appears to stabilize the preformed D2-FGF binary complex.     

Determinants of ligand binding specificity in the glial cell line-derived neurotrophic factor family receptor alpha S

The glial cell line-derived neurotrophic factor (GDNF) family comprise a subclass of cystine-knot superfamily ligands that interact with a multisubunit receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich glycosyl phosphatidylinositol-anchored binding subunit called GDNF family receptor alpha (GFRalpha). All four GDNF family ligands utilize c-Ret as a common signaling receptor, whereas specificity is conferred by differential binding to four distinct GFRalpha homologues. To understand how the different GFRalphas discriminate ligands, we have constructed a large set of chimeric and truncated receptors and analyzed their ligand binding and signaling capabilities. The major determinant of ligand binding was found in the most conserved region of the molecule, a central domain predicted to contain four conserved alpha helices and two beta strands. Distinct hydrophobic and positively charged residues in this central region were required for binding of GFRalpha1 to GDNF. Interaction of GFRalpha1 and GFRalpha2 with GDNF and neurturin required distinct subsegments within this central domain, which allowed the construction of chimeric receptors that responded equally well to both ligands. C-terminal segments adjacent to the central domain are necessary and have modulatory function in ligand binding. In contrast, the N-terminal domain was dispensable without compromising ligand binding specificity. Ligand-independent interaction with c-Ret also resides in the central domain of GFRalpha1, albeit within a distinct and smaller region than that required for ligand binding. Our results indicate that the central region of this class of receptors constitutes a novel binding domain for cystine-knot superfamily ligands.     

N-glycosylation of fibroblast growth factor receptor 1 regulates ligand and heparan sulfate co-receptor binding

The regulation of cell function by fibroblast growth factors (FGF) occurs through a dual receptor system consisting of a receptor-tyrosine kinase, FGFR and the glycosaminoglycan heparan sulfate (HS). Mutations of some potential N-glycosylation sites in human fgfr lead to phenotypes characteristic of receptor overactivation. To establish how N-glycosylation may affect FGFR function, soluble- and membrane-bound recombinant receptors corresponding to the extracellular ligand binding domain of FGFR1-IIIc were produced in Chinese Hamster Ovary cells. Both forms of FGFR1-IIIc were observed to be heavily N-glycosylated and migrated on SDS-PAGE as a series of multiple bands between 50 and 75 kDa, whereas the deglycosylated receptors migrated at 32 kDa, corresponding to the expected molecular weight of the polypeptides. Optical biosensor and quartz crystal microbalance-dissipation binding assays show that the removal of the N-glycans from FGFR1-IIIc caused an increase in the binding of the receptor to FGF-2 and to heparin-derived oligosaccharides, a proxy for cellular HS. This effect is mediated by N-glycosylation reducing the association rate constant of the receptor for FGF-2 and heparin oligosaccharides. N-Glycans were analyzed by mass spectrometry, which demonstrates a predominance of bi- and tri-antennary core-fucosylated complex type structures carrying one, two, and/or three sialic acids. Modeling of such glycan structures on the receptor protein suggests that at least some may be strategically positioned to interfere with interactions of the receptor with FGF ligand and/or the HS co-receptor. Thus, the N-glycans of the receptor represent an additional pathway for the regulation of the activity of FGFs.     

Ligand binding properties of binary complexes of heparin and immunoglobulin-like modules of FGF receptor 2

Epithelial cells, which express FGFR2IIIb, bind and respond to FGF-1, FGF-7 and FGF-10, but not FGF-2. Stromal cells, which bind and respond to FGF-1 and FGF-2, but not FGF-7 and FGF-10, express FGFR2IIIc or FGFR1IIIc. Here we show that when both isolated FGFR2betaIIIb and FGFR2betaIIIc or their common Ig module II are allowed to affinity select heparin from a mixture, the resultant binary complexes bound FGF-1, FGF-2, and FGF-7 with nearly equal affinity. In addition, FGF-2 and FGF-7 bound to both heparin-Ig module IIIb and IIIc complexes, but FGF-1 bound to neither Ig module III. The results show that in isolation both Ig modules II and III of FGFR2 can interact with heparin and that each exhibits a binding site for FGF. We suggest that the specificity of FGFR2IIIb and FGFR2IIIc is dependent on the cell membrane environment and heparin/heparan sulfate. Ig modules II and III cooperate both within monomers and across dimers with cellular heparan sulfates to confer cell type-dependent specificity of the FGFR complex for FGF.     

Fibroblast growth factor receptor-4 shows novel features in genomic structure, ligand binding and signal transduction.

Fibroblast growth factor (FGF) receptor (FGFR) gene family consists of at least four receptor tyrosine kinases that transduce signals important in a variety of developmental and physiological processes related to cell growth and differentiation. Here we have characterized the binding of different FGFs to FGFR-4. Our results establish an FGF binding profile for FGFR-4 with aFGF having the highest affinity, followed by K-FGF/hst-1 and bFGF. In addition, FGF-6 was found to bind to FGFR-4 in ligand competition experiments. Interestingly, the FGFR-4 gene was found to encode only the prototype receptor in a region where both FGFR-1 and FGFR-2 show alternative splicing leading to differences in their ligand binding specificities and to secreted forms of these receptors. Ligands binding to FGFR-4 induced receptor autophosphorylation and phosphorylation of a set of cellular polypeptides, which differed from those phosphorylated in FGFR-1-expressing cells. Specifically, the FGFR-1-expressing cells showed a considerably more extensive tyrosine phosphorylation of PLC-gamma than the FGFR-4-expressing cells. Structural and functional specificity within the FGFR family exemplified by FGFR-4 may help to explain how FGFs perform their diverse functions.     

A novel form of FGF receptor-3 using an alternative exon in the immunoglobulin domain III

Four distinct FGF receptors were cloned and characterized and it was demonstrated that the ligand binding site of FGF receptors is confined to the extracellular immunoglobulin-like (Ig)-domain 2 and 3. The Ig-domain 3 is encoded by two separate exons: exon IIIa encodes the N-terminal half, and the C-terminal half is encoded by either exon IIIb or IIIc in FGFR1 and FGFR2, whereas FGFR4 is devoid of exon IIIb. Alternative usage of exons IIIb and IIIc determine the ligand binding specificity of the receptor. To analyze the arrangement of these exons in FGFR3 we cloned the genomic sequence between exon IIIa and IIIc of FGFR3 and identified an alternative exon, corresponding to exon IIIb of the FGFR1 and FGFR2. The sequence of this exon shows Ig-domain hallmarks, 44% identity with exon IIIb of other FGF receptors and 36% identity with exon IIIc of FGFR3. Using this exon as a probe for mouse RNA as well as PCR analysis, demonstrated that exon IIIb encodes an authentic form of FGFR3 that is expressed in mouse embryo, mouse skin and mouse epidermal keratinocytes. The results demonstrate that the presence of alternative exons for Ig-domain 3 is a general phenomena in FGFR1, 2 and 3, and represents a novel genetic mechanism for the generation of receptor diversity.     

Complexity of FGF receptors: genetic basis for structural diversity and functional specificity.

Since 1989, the receptors for fibroblast growth factors (FGFs) were cloned and characterized as a subgroup of the family of receptor tyrosine kinases. Four FGF receptor genes were identified, all of which encode membrane-bound glycoproteins containing three immunoglobulin (Ig) -like domains at the extracellular region, where only two of these domains are involved in ligand binding. Three unique features characterize the FGF receptors: 1) overlapping recognition and redundant specificity, where one receptor may bind with a similar affinity several of the seven known FGFs and one FGF may bind similarly to several distinct receptors. 2) The binding of FGFs to their receptors is dependent on the interaction of FGF with cell surface heparan sulfate proteoglycans. 3) A multitude of isoforms of cell-bound or secreted receptors are produced by the same gene. The gene structure of these receptors revealed two major mechanisms that are responsible for the formation of the diverse forms: alternative mRNA splicing, resulting in deletions or alternate exons usage, and internal polyadenylation, resulting in truncated products. These are reminiscent of mechanisms that also operate in the immunoglobulin family to generate diversity and to produce either secreted or cell-bound molecules. Tissue-specific alternative splicing in FGF receptors allows for the generation of two distinct receptors from a single gene because alternative exons determine the sequence of the COOH-terminal half of the third Ig-like domain involved in ligand binding. This represents a novel genetic mechanism to generate receptor diversity and specificity and to increase receptor repertoire.     

Model of glycoprotein hormone receptor ligand binding and signaling

Studies described here were initiated to develop a model of glycoprotein hormone receptor structure and function. We found that the region that links the lutropin receptor leucine-rich repeat domain (LRD) to its transmembrane domain (TMD) has substantial roles in ligand binding and signaling, hence we term it the signaling specificity domain (SSD). Theoretical considerations indicated the short SSDs in marmoset lutropin and salmon follitropin receptors have KH domain folds. We assembled models of lutropin, follitropin, and thyrotropin receptors by aligning models of their LRD, TMD, and shortened SSD in a manner that explains how substitutions in follitropin and thyrotropin receptors distant from their apparent ligand binding sites enable them to recognize lutropins. In these models, the SSD is parallel to the concave surface of the LRD and makes extensive contacts with TMD outer loops 1 and 2. The LRD appears to contact TMD outer loop 3 and a few residues in helices 1, 5, 6, and 7. We propose that signaling results from contacts of the ligands with the SSD and LRD that alter the LRD, which then moves TMD helices 6 and 7. The positions of the LRD and SSD support the notion that the receptor can be activated by hormones that dock with these domains in either of two different orientations. This would account for the abilities of some ligands and ligand chimeras to bind multiple receptors and for some receptors to bind multiple ligands. This property of the receptor may have contributed significantly to ligand-receptor co-evolution.