Calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) are related peptides that are potent vasodilators. The CGRP and AM receptors are heteromeric protein complexes comprised of a shared calcitonin receptor-like receptor (CLR) subunit and a variable receptor activity modifying protein (RAMP) subunit. RAMP1 enables CGRP binding whereas RAMP2 confers AM specificity. How RAMPs determine peptide selectivity is unclear and the receptor stoichiometries are a topic of debate with evidence for 1:1, 2:2, and 2:1 CLR:RAMP stoichiometries. Here, we describe bacterial production of recombinant tethered RAMP-CLR extracellular domain (ECD) fusion proteins and biochemical characterization of their peptide binding properties. Tethering the two ECDs ensures complex stability and enforces defined stoichiometry. The RAMP1-CLR ECD fusion purified as a monomer, whereas the RAMP2-CLR ECD fusion purified as a dimer. Both proteins selectively bound their respective peptides with affinities in the low micromolar range. Truncated CGRP(27-37) and AM(37-52) fragments were identified as the minimal ECD complex binding regions. The CGRP C-terminal amide group contributed to, but was not required for, ECD binding, whereas the AM C-terminal amide group was essential for ECD binding. Alanine-scan experiments identified CGRP residues T30, V32, and F37 and AM residues P43, K46, I47, and Y52 as critical for ECD binding. Our results identify CGRP and AM determinants for receptor ECD complex binding and suggest that the CGRP receptor functions as a 1:1 heterodimer. In contrast, the AM receptor may function as a 2:2 dimer of heterodimers, although our results cannot rule out 2:1 or 1:1 stoichiometries.

R-spondins are secreted glycoproteins (RSPO1, -2, -3, and -4) that exhibit proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine rich repeat (LRR)-containing 7-TM receptors LGR4, -5, and -6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present methodology for bacterial expression and purification of the signaling competent, cysteine-rich Fu1-2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1-14, and the human ZNRF3 ECD. In a cell-based signaling assay the non-glycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nM affinities and rank order RSPO4 > RSPO2 > RSPO3 > RSPO1 in a TR-FRET assay. RSPO-receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinity rank order RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and provide a rationale for the stronger signaling potencies of RSPO2 and -3 as resulting from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.

The calcitonin family peptides calcitonin (CT), amylin (AMY), calcitonin gene‐related peptide (CGRP), and adrenomedullin (AM) regulate diverse processes including calcium homeostasis, blood glucose levels, vasodilation, and vascular development. Their receptors are heteromeric cell surface protein complexes comprised of a class B GPCR subunit, either the calcitonin receptor (CTR) or the calcitonin‐like receptor (CLR), in association with a peptide selectivity‐determining receptor activity modifying protein (RAMP) subunit. CTR alone is the CT receptor and CTR complexes with any of three RAMPs are AMY receptors. The CLR‐RAMP1 complex is a CGRP receptor, whereas CLR complexes with either RAMP2 or ‐3 are AM receptors. How RAMPs determine calcitonin family peptide selectivity is unclear and the receptor stoichiometries are a topic of debate. Here, we report unique methods for bacterial production of recombinant CLR‐RAMP extracellular domain (ECD) complexes engineered for structure/function studies and use of the novel constructs for peptide binding studies and crystallization. In addition, we describe recombinant CTR ECD production in HEK293T cells, and production of the integral membrane portions of the receptors in the E. coli inner membrane for structure/function studies. Our results indicate that the CTR and CLR ECDs are sufficient to determine peptide binding selectivity, identify peptide residues critical for receptor binding, shed light on the receptor stoichiometries, and provide methods that will facilitate structural studies of these therapeutically important GPCRs. Grant Funding Source: Supported by NIH grant R01GM104251