The exaggerated bend angle around Pro^6.50, in the CWxPY motif in TM6, is stabilized by a water molecule that makes hydrogen bonds to the Cys^6.47 and Tyr^6.51 residues of the CWxPY motif and to a residue in TM7 in GPCR structures (33, 58, 60–62). The importance of this structure in the GnRH receptor is supported by no less than three cHH-associated mutations of the CWxPY motif. The Pro^6.50(282)Arg mutation of the GnRH receptor completely disrupts receptor function, which cannot be recovered by pharmacoperone treatment (36), showing that the proline kink is essential for GnRH receptor expression. The Cys^6.47(279)Tyr mutant GnRH receptor and a laboratory-produced Cys^6.47(279)Ala mutant showed no measurable GnRH binding and severely decreased cellular responses to GnRH stimulation that were recovered after pharmacoperone treatment of cells transfected with the mutant receptor (1, 35, 63, 64). This suggests that the Cys^6.47(279) mutations disrupt receptor folding during biosynthesis, but the rescue shows that its effect is less destructive than mutating the Pro^6.50(282). A Tyr^6.51(283)His GnRH receptor mutation causes cHH and results in no measurable function in vitro (65).

The overall GPCR fold (the relative positions of the seven TM segments) is stabilized by ~200–260 non-covalent intramolecular contacts (hydrogen bonds, van der Waals interactions, etc.) and by a network of hydrogen-bonded water molecules in the interior of the TM domain. Depending on methodology, 24–40 interhelical contacts between topologically equivalent loci (positions) are present in all active and inactive class A GPCR structures (17, 19, 20). These conserved conformation-independent interhelical contacts determine the overall GPCR structure, forming a conserved “scaffold,” on which conformational changes can occur. Some of the conserved interhelical contacts are required for protein folding and insertion into the membrane during biosynthesis. The conserved interhelical contacts involve many of the residues that are highly conserved in class A GPCRs (Table S1 in Supplementary Material) but also involve residues in topologically equivalent loci, where the amino acids are not conserved (19, 20, 57). Most of the conserved conformation-independent interhelical interactions are located toward the central and cytoplasmic side of the TM domain (20) (Figure S2 in Supplementary Material), consistent with the emerging recognition that these are the areas in which GPCR structure is most conserved, whereas the extracellular side of the TM barrel is less conserved, because of the need to accommodate diverse ligands (18).

Gonadotropin-releasing hormone receptor residues topologically equivalent to the residues in conserved conformation-independent interhelical contacts are listed in Table S1 in Supplementary Material, with the predicted interhelical contacts and effects of previously reported mutations of the residues on GnRH receptor expression and function. Many mutations result in undetectable receptor function, consistent with disruption of cell surface GnRH receptor expression or severe misfolding of the receptor protein. The disruption of functional receptor expression confirms the importance of the residues for GnRH receptor structure and indicates that the conserved interhelical contacts constitute part of the GnRH receptor structure, similar to their roles in other GPCRs.

The complete disruption of GnRH receptor expression when Asn^2.50(87) in TM2 was substituted with Ala or Asp (Table S1 in Supplementary Material) supports a role for the Asn^2.50(87) residue in stabilizing GnRH receptor structure. However, GPCR crystal structures show that Asp^2.50 makes conserved contacts with residues in TM1 and TM7, but it is only connected to Asn^7.49 via the water-mediated hydrogen bond network (19, 58). Based on the conserved structural scaffold, Asn^2.50(87) of the GnRH receptor contacts Asn^1.50(53) and Pro^7.46(316), whereas Asp^7.49(319) does not form any conserved conformation-independent contacts (Table S1 in Supplementary Material).

Many cHH-associated GnRH receptor mutations involve residues that constitute conserved interhelical contacts in the crystallized GPCR structures. These include the Glu^2.53(90)Lys, Glu^2.53(90)Asp, Ala^4.57(171)Thr, Cys^6.47(279)Tyr, Tyr^6.51(283)His, Tyr^6.52(284)Cys, Pro^7.50(320)Arg, and Tyr^7.53(323)Cys mutations. Most cHH-associated mutant receptors are poorly expressed in vitro (Table S1 in Supplementary Material), consistent with the mutations disrupting the structural scaffold of the receptor. Disruption of interhelical contacts would destabilize receptor protein folding, resulting in fewer correctly folded receptor molecules being transported to the cell membrane or decreased residence time of less stable receptor proteins once they get to the cell membrane. Pharmacoperones act as templates for folding of nascent receptor proteins, stabilizing biosynthesis, and increasing protein expression (34, 35, 47). In most cases, pharmacoperone treatment of cells transfected with cHH-associated mutant GnRH receptors, enhanced mutant receptor expression (Table S1 in Supplementary Material) and the “rescued” receptors showed wild type-like function. This supports a role for the mutated residues in stable folding and cell surface expression of the GnRH receptor (34, 35, 47).

The late Michael Conn’s laboratory and others proposed that the Glu^2.53(90) side chain of the GnRH receptor forms a interhelical salt-bridge with Lys^3.32(121) in TM3 and that the cHH-associated Glu^2.53(90)Lys mutation disrupted receptor biogenesis and folding by breaking this salt bridge (66–69). Although Glu^2.53(90) is conserved in type 1 GnRH receptors, type 2 GnRH receptors and other class A GPCRs have large hydrophobic residues at position 2.53 (13, 43, 49, 57). The lack of conservation suggests that the acidic side chain of Glu^2.53(90) may not be necessary for type 1 GnRH receptor structure and that the effect of the Glu^2.53(90)Lys mutation may result from disruptive effects of introducing Lys, rather than lack of Glu. This is supported by a mutation of Glu^2.53(90) to uncharged Gln, which had no effect on receptor function (70) and a conservative Glu^2.53(90)Asp, which is associated with cHH (Table S1 in Supplementary Material). A Glu^2.53(90)Ala mutation resulted in undetectable GnRH receptor function (68), but we have not found any report of the effect of the Glu^2.53(90)Asp mutation, which would formally test the salt bridge hypothesis.

The conserved interhelical contacts in GPCR structures predict that Glu^2.53(90) interacts with Ser^3.35(124) (Table S1 in Supplementary Material). Interaction of Glu^2.53(90) with Ser^3.35(124) is supported by an automated (unbiased) structural homology model of the GnRH receptor (71), which shows Glu^2.53(90) close to Ser^3.35(124), whereas Lys^3.32(121) points away, toward Asp^2.61(98) (Figure 1). A Ser^3.35(124)Asp mutation resulted in undetectable binding, suggesting that the mutation caused receptor instability by introducing close apposition of two carboxyl side chains (37). Thus, it is likely that the cHH-associated Glu^2.53(90)Lys mutation disrupts GnRH receptor expression by disrupting the conserved interhelical contact with the 3.35 locus, rather than disrupting a salt-bridge with Lys^3.32(121).

Another cHH-associated GnRH receptor mutant, Tyr^6.51(283)His, also showed undetectable function in vitro (65). Since Tyr^6.51 forms a conserved interhelical contact with the residue in the 7.39 locus, the Tyr^6.51(283)His mutation may disrupt receptor structure by disrupting an interhelical contact of Tyr^6.51(283) with Phe^7.39(309) in TM7 of the GnRH receptor (Table S1 in Supplementary Material).

The Trp^6.48(280) residue in the CWxPY motif was proposed to directly contact the Trp³ residue of the GnRH peptide (69, 73). Systematic mutagenesis of Trp^6.48(280) to Ala, His, Ser, Gln, and Met disrupted GnRH receptor expression, as did mutation of Trp^6.48(279) of the rat GnRH receptor to Arg or Ser (74). However, once expression of the mutant receptors was recovered, using a pharmacoperone, the mutant receptors displayed unchanged ligand-binding affinity and signaling (37, 38, 73). This shows that Trp^6.48(280) does not directly contact GnRH, but is important for GnRH receptor structure. Trp^6.48(280) likely forms conserved interhelical contacts with Met^3.36(125) and Ala^7.42(312) of the GnRH receptor (Table S1 in Supplementary Material), which would be disrupted by the mutations.

The inactive rhodopsin structure showed a salt bridge between Arg^3.50 of the D/ERY motif at the cytoplasmic end of TM3, and Glu^6.30 at the cytosolic end of TM6 (28, 82). This “ionic-lock” interaction stabilizes the inactive GPCR conformation by drawing the cytoplasmic ends of TM3 and TM6 together (26, 83, 84). However, the salt bridge between Arg^3.50 and Glu^6.30 cannot provide a universal mechanism for stabilizing inactive GPCR conformations, because Glu^6.30 is not conserved (83). The GnRH receptor has Arg^6.30(262), which clearly cannot form a salt bridge with Arg^3.50(139). Nevertheless, mutations show that both Arg residues are important for GnRH receptor structure and function. The cHH-associated Arg^6.30(262)Gln mutation decreased ligand binding and cellular signaling (85), which was recovered when cells were treated with pharmacoperone (1, 47), suggesting that the Arg^6.30(262) side chain forms an intramolecular interaction that stabilizes folding of the unoccupied GnRH receptor. Since GPCRs are biosynthesized in cellular compartments that are inaccessible to endogenous ligands, receptors are likely synthesized in inactive conformations. Inactive conformation-specific interhelical contacts may thus stabilize receptor biosynthesis, so mutations that disrupt these contacts may also disrupt expression.

Mutation of Arg^3.50(139) in the DRY/S motif to other basic amino acids, His or Lys, resulted in no measurable receptor function, consistent with disruption of a structurally important interaction. An Arg^3.50(139)Gln mutation increased GnRH receptor expression and decreased activation of cellular signaling (29). This suggests that the Gln side chain mimics an interhelical interaction of Arg^3.50(139) that stabilizes receptor folding and stabilizes the inactive receptor conformation. The importance of the Arg^3.50(139) side chain for GnRH receptor structure is also supported by cHH-associated Arg^3.50(139)His and Arg^3.50(139)Cys mutations, which are poorly expressed (30, 78), but rescued by pharmacoperone treatment (Table 2) suggesting that the mutations disrupt biosynthesis of the unoccupied (inactive) GnRH receptor. Since Arg^3.50(139) cannot form an ionic lock in the GnRH receptor, it must form a different contact. GPCR structures show a conserved inactive conformation-specific interhelical contact of Arg^3.50 with the 6.37 locus (19). Thus, the Arg^3.50(139) side chain may stabilize the inactive GnRH receptor conformation by forming an interhelical hydrogen bond with Thr^6.37(269) (Table 2), which may be enhanced in the Arg^3.50(139)Gln mutant receptor.

Many conserved inactive conformation-specific interhelical contacts involve residues in TM3 and TM6 (19), suggesting that, like the ionic lock, they maintain close proximity of the cytoplasmic ends of TM3 and TM6. Met^3.43(132), conserved as a large hydrophobic residue in most GPCRs, is likely to stabilize the inactive GnRH receptor conformation by interacting with Phe^6.40(272) and Ala^6.41(273) in TM6 (Table 2). Mutation of Met^3.43(132) and four mutations of Phe^6.40(272) decreased GnRH receptor expression, suggesting that they disrupt receptor biogenesis. In contrast, the Phe^6.40(272)Leu mutation increased receptor expression (Table 2; Figure S3 in Supplementary Material) (77, 81), suggesting that the Phe^6.40(272)Leu mutation enhances the TM3-TM6 interhelical contact with Met^3.43(132) and so enhances expression of the inactive GnRH receptor.

A more recent study, using five pairs of GPCR structures found that only one TM3–TM6 inactive conformation-specific contact is conserved (17). This interaction between the 3.46 and 6.37 loci is the key contact that defines the “closed” conformation of the G protein-binding site and prevents G protein binding (17). In the GnRH receptor, these residues are Ile^3.46(135) and Thr^6.37(269) (Table 2). Mutation of Ile^3.46(135) to Ala and Val both completely ablated GnRH receptor function (29). In the context of the inactive GPCR crystal structures, the Ala and Val side chains are likely too short to “fulfill the distance criteria for contact formation” (17). Substitution of Ile^3.46(135) with Leu, which has a large branched aliphatic side chain, partially preserved GnRH receptor expression and increased coupling efficiency (29). Thus, the Ile^3.46(135)Leu mutation may destabilize the inactive conformation of the GnRH receptor by weakening the Ile^3.46(135)–Thr^6.37(269) interhelical contact and favoring formation of the R* active conformation. A cHH-associated Thr^6.37(269)Met mutation resulted in no measurable function in several assay systems (80). This result confirms the importance of Thr^6.37(269) in GnRH receptor structure, but provides no information about its function in receptor conformation.

The second key inactive conformation-specific interhelical contact orients Tyr^7.53 of the NPxxY motif in TM7 toward TM1, where it contacts the residue in the 1.53 locus, keeping the Tyr^7.53 side chain away from the interior of the TM domain (17, 19). The corresponding residues of the GnRH receptor are Phe^1.53(56) in TM1 and Tyr^7.53(323) in the N/DPxxY motif in TM7 (Table 2).

Higher resolution GPCR structures revealed internal water molecules that form a conserved network, with hydrogen bonds connecting the conserved amino acids in different helices, including Asn^1.50, Asp^2.50, Trp^6.48 of the CWxPY motif and residues of the NPxxY motif. The water molecules both stabilize the GPCR structural fold and mediate transition between conformational states, by forming many “low energy switches,” which can be broken and reconfigured during GPCR activation (22, 26, 33, 50, 86). The GnRH receptor likely has a network of intramolecular water molecules between its highly conserved amino acids and mutations that disrupt GnRH receptor expression or function, may do so by disrupting the water-mediated intramolecular network.

The hydrophilic network in inactive GPCRs also includes a sodium ion, which makes a conserved contact with Asp^2.50 and stabilizes the inactive receptor conformation. Cations decrease GnRH receptor agonist binding (87). Since type 1 GnRH receptors do not have Asp^2.50, the nearby Glu^2.53(90) and Asp^7.49(319) (of the N/DPxxY motif) residues may provide negative charges that enhance cation binding, as suggested for the PAR1 receptor (22, 24, 88, 89).

All inactive class A GPCR structures have a layer of hydrophobic amino acids on the cytosolic side of Asp^2.50 that separates the water molecules in the ligand- and G protein-binding pockets. The barrier stabilizes the inactive GPCR conformation and consists of conserved class A GPCR residues, including some that also form conserved inactive conformation-specific interhelical contacts (26, 33, 75). The partially overlapping “central hydrophobic core,” consisting of the conserved Phe^6.44 and conserved hydrophobic residues in the 3.43 and 6.40 loci (90), the “core triad” consisting of Phe^6.44, Pro^5.50 and the hydrophobic residue at position 3.40 (22) and the “transmission switch,” consisting of the hydrophobic residue at position 3.40, Pro^5.50, Leu^5.51, and Phe ^6.44, which is one helical turn away from Trp^6.48 of CWxPY motif, all couple the ligand-binding pocket to the hydrophobic barrier (26, 31, 32). The GnRH receptor has hydrophobic residues in the loci associated with the hydrophobic barrier and the transmission switch, so it is likely that the GnRH receptor has a hydrophobic barrier that stabilizes its inactive conformation.

In spite of the diversity of ligand-binding surfaces many ligands contact highly conserved residues, particularly Trp^6.48 and Tyr^6.51 of the CWxPY motif. In the active structure of rhodopsin retinal isomerization induces movement of Trp^6.48, which causes rotation of TM6, without changing either the rotamer angle of the Trp^6.48 side chain or the hinge angle of the proline kink. The exaggerated bend angle of Pro^6.50 amplifies movement of the cytoplasmic end of TM6, which moves outward, away from TM3 (33). Similar rotation of the cytoplasmic end of TM6 was seen in all fully active GPCR structures (22, 27, 56, 92, 93).

Rotation of TM6 also changes its interhelical contacts with TM3 and TM5. The Phe^6.44 side chain moves toward TM5, where it rearranges the Leu^5.51 and Pro^5.50 residues and moves the conserved Leu^3.40 of TM3 away from TM5, thus triggering the “transmission switch” (26, 31). Similar activation of the transmission switch via agonist-induced movement of Trp^6.48 is seen in the A_2A-adenosine and μ-opioid receptors (22, 31). The transmission switch changes the conformation of the proline kink of TM5 and rotates the conserved Tyr^5.58 side chain (near the cytoplasmic end) inwards. It also changes the position of TM3, rotating it and moving it slightly toward the extracellular of side the TM domain (90).

Active structures of other GPCRs showed similar outward movement of the cytosolic end of TM6 but showed no agonist contact with Trp^6.48 (31, 93–98), so TM6 movement must be achieved via different mechanisms. In the β₂-adrenergic receptor, agonist binding at the extracellular end of TM5 causes movement of Pro^5.50, which moves Ile^3.40 in TM3 and Phe^6.44 in TM6, thus opening the core triad, triggering the transmission switch and rotating the cytoplasmic end of TM6 away from the helix bundle (26, 31). A similar opening of the core triad residues occurs in the μ-opioid receptor, except that the agonist binds to two residues (the 3.32 and 3.36 loci) in TM3, resulting in movement of Ile^3.40, which activates the core triad (22). Mutation of the Lys^3.32(121) residue in TM3 of the GnRH receptor decreased binding of GnRH agonists but not antagonists (99), suggesting that GnRH interaction with Lys^3.32(121) initiates activation. Although mutagenesis experiments do not support a role for Trp^6.48(280) in ligand binding or activation of the GnRH receptor (38), there is evidence that GnRH contacts Tyr^6.58(290) at the extracellular end of TM6 (100) and that Tyr^6.51(283) of the CWxPY affects ligand-binding affinity (37), suggesting that these residues may initiate rotation of TM6.

The water-mediated polar network in active GPCR structures differs from that of inactive structures. Agonist-induced movement of the extracellular ends of the TM helices rearranges water molecules at the extracellular side of the TM domain and opens the hydrophobic barrier, which allows formation of a continuous water channel between the ligand-binding pocket and the cytoplasmic surface of the receptor. This changes the conformation of TM7 and causes rotation of Tyr^7.53 away from its interaction with TM1 toward the center of the TM bundle (22, 32, 33, 50, 75, 90, 96, 101, 102). The rearranged water molecules link Arg^3.50 with Tyr^5.58 and Tyr^7.53 in a water-mediated interhelical network that can be considered the “open” conformation of the ionic lock that stabilizes the active receptor conformation (22, 28, 96). Movements of TM6 and TM7 collapse the sodium ion-binding pocket, making it too small to accommodate the ion (24, 88, 97) and the cation moves toward the cytoplasm through the open hydrophobic barrier (24). Mutation of the water-associated Arg^3.50(139), Asp^7.49(318) and Tyr^7.53(322) residues decreases GnRH receptor coupling efficiency (23, 29, 30, 39, 77, 103) indicating that they have roles in the active receptor conformation, which may be mediated by the water network.

Rotation of TM6 and opening of the hydrophobic barrier break the inactive conformation-specific interhelical contacts and form new active conformation-specific interhelical contacts. The ionic lock opens and the Arg^3.50 side chain moves into the space vacated by TM6 where it orients toward Tyr^5.58 of TM5. Arg^3.50 and Tyr^5.58 form new interhelical contacts with the hydrophobic residue in position 6.40 (19, 22, 28). The GnRH receptor has Asn^5.58(231), which is smaller than Tyr. The NTSR1 neurotensin receptor also has Asn^5.58 and an “active-like” NTSR1 structure shows a hydrogen bond between Asn^5.58(257) and Arg^3.50(167) (104), which suggests that Asn^5.58(231) stabilizes the open ionic lock in the GnRH receptor.

On activation Met^3.43 breaks its inactive conformation-specific interhelical contact with the residues in positions 6.40 and 6.41. The release of the hydrophobic side chain in position 6.41 allows it to contact the hydrophobic residue in position 5.55, forming one of two key active conformation-specific interhelical contacts (17, 19). In the GnRH receptor these residues are Leu^5.55(228) and Ala^6.41(273) (Table 2). One helical turn closer to the cytoplasmic side of the receptor, the Ile^3.46 side chain breaks its inactive conformation-specific contact with the position 6.37 side chain and forms a new interhelical contact with Tyr^7.53, forming the second key active conformation-specific interaction (17, 19). The breaking of the TM3-TM6 contacts opens a cleft that allows G protein access and releases the position 6.37 residue to make a conserved interaction with the G protein (17, 27) (Figure S3 in Supplementary Material). In the GnRH receptor these residues are Ile^3.46(135), Thr^6.37(269) and Tyr^7.53(323) (Table 2). Mutation of Ile^3.46(135) to Leu increased GnRH receptor coupling efficiency (29), suggesting that the Leu side chain may favor interaction with Tyr^7.53(323) over interaction with Thr^6.37(269), thus favoring the active conformation. A role for Tyr^7.53(323) in stabilizing the active GnRH receptor conformation is supported by the Tyr^7.53(323)Ala mutant, which did not activate cellular signaling (39, 77).

The endothelin-1 peptide penetrates the TM core of the ET_B-endothelin receptor and contacts the consensus-binding residues, Trp^6.48 and Leu^6.51 and Gln^3.32 (107). The smaller peptide agonist NTS_8–13 binds closer to the extracellular surface of the NTSR1 neurotensin receptor, but may contact the consensus Arg^3.32(149) residue (104). Mutation of the equivalent GnRH receptor residue, Lys^3.32(121), to Gln or Ala decreased GnRH affinity and signaling, but had minimal effect on binding of a peptide antagonist, which had modified amino-terminal residues. This led to a conclusion that Lys^3.32(121) may form a hydrogen bond with the aromatic rings of His² or Trp³ of GnRH (37, 99). Subsequent models proposed that Lys^3.32(121) contacts pGlu¹ or His² (37, 68, 110–113) but, in the absence of further experiments, it remains uncertain whether Lys^3.32(121) directly contacts GnRH or initiates receptor activation.

Agonist peptide ligands bound to the NTSR1 neurotensin, US28 viral chemokine and apelin receptors do not penetrate deeply enough to contact Trp^6.48 of the CWxPY motif (93, 104, 106) and mutagenesis of Trp^6.48 had minimal effects on NTSR1 receptor function (114). Molecular models suggested that Trp^6.48(280) of the GnRH receptor interacts with Trp³ of the GnRH peptide (13, 69, 74, 111). However, mutations of Trp^6.48(280) had minimal effects on GnRH affinity or cellular signaling (37, 38), indicating that it does not directly contact GnRH. Since TM3 has central roles in ligand binding, the conserved interhelical network and the hydrophobic core, movement of TM3 may provide an alternative molecular pathway to Trp^6.48-mediated activation of the transmission switch in the GnRH and NTSR1 receptors (20, 57).

The hydrophobic residue in position 6.51 contacts the ligand in the ET_B-endothelin and apelin receptors (106, 107), whereas the conserved Glu^7.39 of chemokine receptors is a key determinant of chemokine binding and receptor activation (109). A Tyr^6.51(283)Phe mutation in the GnRH receptor and mutations of Phe^7.39(309) to Leu or Gln decreased GnRH-binding affinity (37). A recent computational model suggests that Phe^7.39(309) may contact Trp³ of GnRH (110). Together, these data are consistent with the mutations disrupting ligand binding by breaking an interhelical contact between residues that may also contact the ligand, but more experiments are needed.

In summary, it is possible that amino-terminal residues of the GnRH peptide contact some of the consensus ligand-binding residues, Lys^3.32(121), Tyr^6.51(283), and Phe^7.39(309), but not Trp^6.48(280). It remains uncertain whether GnRH binds to a largely extracellular surface of the receptor like NTS_8–13 (104) or penetrates the TM core like the peptide ligands of the apelin and chemokine receptors (106, 109).

A molecular model predicted that Arg^1.35(38) in the amino terminus of the GnRH receptor is close to the carboxy-terminal Pro⁹-Gly¹⁰NH₂ of GnRH. Mutations of Arg^1.35(38) decreased GnRH-binding affinity, but had lesser effects on binding of [Pro⁹-NHEt]-GnRH, which lacks Gly¹⁰NH₂ (112). The results support a hydrogen bond contact between Arg^1.35(38) and Gly¹⁰NH₂, but show that both the geometry and charge of the Arg^1.35(38) side chain are important for additional inter- or intramolecular interactions. Asn^2.65(102) in TM2 has similar functions in distinguishing Gly¹⁰NH₂ of GnRH (13, 115). These studies suggest that the carboxy-terminus of GnRH may locate close to both Arg^1.35(38) and Asn^2.65(102) (Figure S4 in Supplementary Material). Systematic mutagenesis of Asp^2.61(98) of the GnRH receptor, combined with ligand modification, showed that the Asp^2.61(98) side chain determines receptor recognition of His² of the GnRH peptide, via a hydrogen bond, whereas the charge of the Asp^2.61(98) side chain may configure the surface of the ligand-binding pocket by forming an interhelical salt bridge with Lys^3.32(121) (13, 116). Thus, residues in the amino-terminus and extracellular ends of TM1 and TM2 of the GnRH receptor appear to affect GnRH binding via direct contacts with amino- and carboxy-terminal residues of the peptide and via intramolecular interactions that affect the shape of the ligand-binding surface.

Molecular models of the GnRH receptor showed contact of Tyr^6.58(290), two helical turns toward the extracellular end of TM6 from the CWxPY motif, with the Tyr⁵ side chain of the GnRH peptide. Systematic mutagenesis showed that both the hydroxyl group and the aromatic ring of the Tyr^6.58(290) side chain contribute to high affinity binding of GnRH, but had less effect on binding of [Ala⁵]-GnRH, consistent with the hydroxyl group of Tyr^6.58(290) interacting with the aromatic ring of Tyr⁵ of the peptide. The receptor mutations also decreased GnRH potency in signaling assays more than they decreased binding affinity, showing that the Tyr^6.58(290) side chain has an additional role in coupling agonist binding to receptor activation (100). The Tyr^6.58(290)–Tyr⁵ interaction may initiate movement and rotation of TM6 in the GnRH receptor.

Mutation of His^7.36(305) at the extracellular end of TM7 in the mouse GnRH receptor to non-polar amino acids decreased GnRH-binding affinity, suggesting loss of a hydrogen bond interaction. Ligand modification suggested a His^7.36(305)-Trp³ hydrogen bond contact. However, mutation of His^7.36(305) to polar amino acids had no effect on ligand-binding affinity, making a direct interaction with the ligand unlikely. Molecular modeling showed that His^7.36(305) made only intramolecular contacts with the amino terminus of the receptor, whereas Trp³ of GnRH was oriented near the consensus ligand-binding residue, Phe^7.39(308). This suggests that His^7.36(305) forms an interhelical contact that positions Phe^7.39(308) to form π–π contact with Trp³ of the peptide (110). The cHH-associated GnRH receptor mutation, Thr³²Ile, which decreases ligand-binding affinity (63), is immediately adjacent to the His^7.36(305) interhelical contacts and may disrupt the interhelical contact.

Mammalian GnRH has a basic Arg⁸ residue, which is important for binding to type 1 GnRH receptors. Mutation of the acidic Asp^7.32(302) residue to uncharged Asn decreased binding affinity of GnRH, but had no effect on binding of peptides with uncharged Gln⁸. This suggests that Asp^7.32(302) forms a salt bridge contact with Arg⁸ of the peptide. However, “conformationally constrained” GnRH peptides, in which the high affinity β-turn was stabilized by a d-amino acid in position 6, retained high affinity binding in the absence of Asp^7.32(302) or Arg⁸ or both. Since the Arg⁸ side chain also contributes to stabilizing the β-turn in the native GnRH peptide, it was concluded that the interaction of Asp^7.32(302) with Arg⁸ induces the high-affinity peptide conformation (70, 117).