Class B G-protein-coupled receptors (GPCRs) are key players in hormonal homeostasis and important drug targets for endocrinal and neuronal disorders. Growth hormone-releasing hormone receptor (GHRHR), a prototypical class B GPCR, is expressed by somatotropic cells of the pituitary gland. Activation of GHRHR by GHRH, a 44-amino acid peptide released by the hypothalamus, results in the secretion and production of growth hormone (GH) through cyclic adenosine monophosphate (cAMP)-dependent pathways. Numerous studies demonstrated that GHRH exerts a variety of bioactivities due to its wide distribution and autocrine/paracrine mechanisms. Therefore, GHRH and its analogs, including tesamorelin, MR-409, JI-38, and MIA-690, have been developed as potential therapeutic agents to treat diabetes, cancers, and cardiovascular diseases.
Like other class B GPCRs, GHRHR consists of an extracellular domain (ECD) and a seven-transmembrane helix domain (7-TMD). Recently published cryo-electron microscopy (cryo-EM) structures of class B GPCRs bound to a G s heterotrimer protein include parathyroid hormone receptor 1 (PTH1R), glucagon-like peptide-1 receptor (GLP-1R), calcitonin receptor, calcitonin gene-related peptide receptor(CGRPR), two subtypes of corticotrophin-releasing factor (CRF1R and CRF2R), and adrenomedullin receptors(AM1R and AM2R), as well as pituitary adenylate cyclase-activating polypeptide(PACAP) type I receptor(PAC1R) and vasoactive intestinal polypeptide receptor (VIP1R), revealing a common mode of ligand-induced receptor activation. The C-terminal α helix of peptide ligand recognizes and binds to the ECD, thereby allowing its N-terminus to interact with the extracellular TM core. This is followed by a major conformational change that involves a large kink at the TM6 to open the intracellular face for G protein coupling. However, ligand-binding specificity and roles of ECD in receptor activation vary widely among class B GPCRs due to diverse amino acid sequences of both peptidic ligands and receptors.
Here, we employed the single-particle cryo-EM approach to determine the near-atomic resolution structure of the human GHRHR bound to GHRH in complex with a heterotrimeric G s protein. Together with functional studies and molecular dynamics (MD) simulations, our results provide key insights into the structural basis of ligand recognition, receptor activation, and isolated growth hormone deficiency (IGHD) causing mechanism related to GHRHR, thereby offering a template for rational design of drugs against this receptor.
We developed a NanoBiT tethering strategy to stabilize the assembly of a GHRH–GHRHR–G s complex for cryo-EM studies, overcoming the lack of stability of the above complex (Supplementary Fig.1 and Supplementary Table1), as it has been used for the VIP1R-G s complex. Using this approach, we were able to obtain a GHRH-GHRHR–G s complex with improved homogeneity and stability (Supplementary Fig.2). The GHRH–GHRHR–G s complex was vitrified and cryo-EM images were collected under a Titan Krios microscope equipped with K2 summit direct detector. The structure of GHRH–GHRHR–G s complex was determined from 307,018 particles to an overall resolution of 2.6 Å (Supplementary Figs.3 and 4). The resulting model contains 28 residues of GHRH (residues 1–28), Gαβγ subunits except the α-helical domain (AHD) of Gα s, and GHRHR residues from 119 to 394. Besides, the ECD region of GHRHR was not resolvable with this limited dataset, perhaps reflecting its highly dynamic and conformationally flexible property when bound to GHRH. We rigid-body fitted the GHRHR ECD (residues 25–118) crystal structure (PDB accession: 2XDG) to the low-pass filtered map (Fig.1b). The majority of amino acid side chains were well resolved in the final model (Supplementary Fig.5), which was refined against the EM density map (Fig.1a) with excellent geometry (Supplementary Table2). Owing to the high-resolution map, we identified one water molecule in the orthosteric binding site, and two water molecules in the G protein engaging pocket. Akin to cryo-EM structure of PTH1R–G s complex, the TMD of GHRHR is surrounded by annular detergent micelle with a diameter of 10 nm, mimicking the natural phospholipid bilayer. Within the micelle, one bound cholesterol and two lipids are also clearly visible in the cryo-EM map.
Fig. 1: The overall cryo-EM structure of GHRH–GHRHR–G s complex.
a Cryo-EM density map that illustrates the GHRH–GHRHR–G s complex and the disc-shaped micelle. The unsharpened cryo-EM density map at the 0.005 threshold shown as light gray surface indicates a micelle diameter of 10 nm. The colored cryo-EM density map is shown at 0.028 threshold. b GHRH–GHRHR–G s complex model and GHRHR ECD crystal structure model docked into the cryo-EM map. c Cartoon representation of the GHRH–GHRHR–G s complex is shown with annular lipids in purple stick representation. Lime green, GHRHR; blue, GHRH; yellow, G s Ras-like domain; red, Gβ; orange, Gγ; gray, Nb35; plum, lipid, and cholesterol.
In the complex structure, GHRH adopts an α-helical configuration when engaged with GHRHR. Compared to GLP-1 (ref. 10) and long-acting parathyroid hormone analog (LA-PTH), GHRH binds to the GHRHR TMD through a more extensive and continuous network of interactions involving all the extracellular loops (ECLs), all the TM helices except TM4, and the linker connecting ECD and TMD. The N-terminus of GHRH inserts deeply into the TMD core and engages an extensive set of receptor-specific interactions (Fig.2a–c and Supplementary Table3). Interestingly, Tyr 1P locates in the equivalent position of the second residue in other class B GPCR peptidic agonists, such as exendin-P5 (ExP5) and LA-PTH, where their first residues of the side chain have different orientations (Fig.2b). The hydroxyl group of Tyr 1P forms hydrogen bonds with H210 3.37b (class B GPCR numbering in superscript) and van der Waals interactions with T213 3.40b and W282 5.36b, whereas the main chain NH of Tyr 1P forms hydrogen bond with R357 7.38b of TM7 and contacts a water molecule that connects with N346 6.57b of TM6-ECL3 hinge, possibly stabilizing the ECL3 in an active state. This observation is consistent with our observation that impairing these contacts dramatically decreased the potency of GHRH in stimulating cAMP accumulation (Fig.2d–f and Supplementary Table4). The most conserved Asp/Glu at position 3 across peptide hormones of the glucagon receptor (GCGR) subfamily, Asp 3P in the case of GHRH, forms salt bridges with K182 2.67b, which is further strengthened by a polar network composed of Thr 7P, Y133 1.43b, and D183 2.68b. From evolutional biology perspective, both Asp 3P and K182 2.67b are fully conserved for GHRH and GHRHR from dozens of species (Supplementary Fig.6a–b). Indeed, D 3P A and K182 2.67b A diminished the potency of GHRH by ~4- and 200-fold (Fig.2e and Supplementary Fig.6c), respectively.
