The GPS motif is a molecular switch for bimodal activities of adhesion class G protein-coupled receptors

Abstract

Adhesion class G protein-coupled receptors (aGPCR) form the second largest group of seven-transmembrane-spanning (7TM) receptors whose molecular layout and function differ from canonical 7TM receptors. Despite their essential roles in immunity, tumorigenesis, and development, the mechanisms of aGPCR activation and signal transduction have remained obscure to date. Here, we use a transgenic assay to define the protein domains required in vivo for the activity of the prototypical aGPCR LAT-1/Latrophilin in Caenorhabditis elegans. We show that the GPCR proteolytic site (GPS) motif, the molecular hallmark feature of the entire aGPCR class, is essential for LAT-1 signaling serving in two different activity modes of the receptor. Surprisingly, neither mode requires cleavage but presence of the GPS, which relays interactions with at least two different partners. Our work thus uncovers the versatile nature of aGPCR activity in molecular detail and places the GPS motif in a central position for diverse protein-protein interactions.

Graphical abstract

Figure 1

7TM Families and Fertilization Defects of lat-1 Mutants (A) Confirmed and putative (indicated by question marks) sites of ligand interaction for the five 7TM receptor families. See also Figure S1. (B) Gonads of a wild-type N2 and lat-1 mutant hermaphrodites; (a) oocytes (O) pass through the spermatheca (arrowhead), become fertilized and embryogenesis ensues. Embryos (E) are visible in the uterus after spermatheca passage. (b,c) lat-1(ok1465) mutant hermaphrodite uteri contain a significant fraction of unfertilized oocytes, whereas oocytes in the gonads appear stacked. (d) The fertility defect of lat-1(ok1465) hermaphrodites can be rescued by a ΔTM2-7 transgene and by wild-type sperm from mating with wild-type males. Scale bars represent 10 μm. (C) Quantification of unfertilized oocytes from different genotypes. Experimental error indicated as SEM.

Figure 2

Transgenic Rescue of lat-1 Mutants Uncover Different Domain Requirements of the LAT-1 Receptor (A–C) LAT-1 receptor domain modifications exhibit different capacities to rescue lethality (circles) and fertility (boxes) of lat-1(ok1465) mutants. Note that the increased number of adult survivors in some transgenic lines is mainly due to increased numbers of eggs laid, whereas these constructs have little effect on the relative rate of developmental failure (gray circles). Previously published constructs are included for comparison and marked by asterisks. For numbers and detailed statistical analysis see Tables S1 and S4. (D) Deconvolved fluorescence image showing LAT-1::GFP expression at the plasma membrane of pharyngeal muscle cells (arrowheads), neurons in the nerve ring and the pharyngeal nervous system (arrows). (E) A ΔHRM variant exhibits an expression pattern indistinguishable from the control fusion protein in (D). Scale bars represent 10 μm. See also Figures S2 and S3.

Figure 3

The HRM of the LAT-1 Receptor Is Not Required for Ligand Binding (A) B1/secretin-type HRM aligned to representative HRM of aGPCR. The Cys residue in position 58 of the alignment is absent in aGPCR HRM (arrowhead). Residues forming the N-terminal α helix (a) and ligand binding pocket (b) in GLP1R are boxed. Disulfide bridges are numbered 1–3 (orange), residues that form the ligand binding interface are labeled in cyan and correspond to structures in (B) and (C). Species abbreviations: ce, Caenorhabditis elegans; dm, Drosophila melanogaster; hs, Homo sapiens; mb, Monosiga brevicollis. (B and C) Orthogonal views of 3D structures of the HRM of human GLP1R (B) (Runge et al., 2008) and a structural model of the C. elegans LAT-1 HRM (C) derived from GLP1R data. The N-terminal α helix (a) and the extended loop (b) are boxed in the GLP1R structure (B) whereas they are both absent in the LAT-1A HRM (C).

Figure 4

Structure but Not Autocatalytic Function of the GPS Is Required for LAT-1 Activity and Expression (A) Western blot of a ΔTM2-7::GFP fusion protein extracted from transgenic worm culture (N2: wild-type C. elegans control) show that the receptor is cleaved. (B) Point mutations H528A and T530A abolish cleavage activity. Full-length (arrows) and cleaved proteins (arrowheads). See also Figure S4. (C and D) Transgenically expressed modified LAT-1::GFP transgene products are delivered to the membrane in neurons (arrows) and pharyngeal muscle cells (arrowheads) independent of GPS cleavage (C) or exchange of the LAT-1 GPS for the LAT-2 GPS. See also Figures S2 and S3. For domain nomenclature see also legend in Figure 2. (E) Deletion of the GPS and a missense mutation (C497S), which abolishes its structural integrity, abrogate rescuing activity of respective full-length transgenes. In contrast, mutations that leave the GPS structure unaffected possess wild-type rescue function. A heterologous GPS from LAT-2 within the LAT-1 receptor context rescues fertility but not developmental phenotypes of lat-1(ok1465) mutants. (F) The remaining rescuing function of the LAT-1/LAT-2 chimeric receptor relies on the presence of the 7TM/C terminus module but not its autocatalytic activity. For domain nomenclature see also legend in Figure 2 and Tables S1 and S4.

Figure 5

Signaling of the LAT-1 Receptor via Cross-Activation in Dimeric Complex (A) Intermolecular complementation of the lat-1(ok1465) phenotype is fully achieved by pairs of donor (left) and recipient (right) LAT-1 receptors independently of GPS cleavage. The RBL domain is required in both partners. See also Tables S1 and S4. (B) Analytical ultracentrifugation of LAT-1 ectodomain fractions. The N terminus contained a T530A GPS point mutation disabling cleavage but not impairing function. The receptor can adopt monomeric (left panel) and a tight dimeric form (right panel). The predicted masses of 60 kDa for a monomer and 120 kDa for a dimer were obtained from protein samples at 20°C using sedimentation equilibrium measurements. (C) A nonreducing polyacrylamide gel reveals a single band (60 kDa) in both monomer and dimer samples indicating that the dimeric form of the LAT-1 N terminus is not covalently linked. Additional bands smaller in size than 60 kDa indicate degraded protein.

Figure 6

Genetic Interaction and Coexpression of lat-1 and ten-1 (A and B) Frequency of developmental (A) and fertility (B) defects in animals with different dosages of lat-1 and ten-1. Genotypes ordered in ascending severity of phenotype. Experimental error indicated as SEM. See also Table S3. (C and D) Expression of lat-1::gfp (C) and ten-1a::gfp (D) transgenes in epidermoblasts derived from the Caaa lineage during dorsal intercalation. Note that in Cpaaaa only ten-1a::gfp expression was found. Scale bars represent 10 μm. (E) Summary of lat-1::gfp and ten-1a::gfp activity in Caaax and Cpaaax lineages. Expressing cell, filled bullet; nonexpressing cell, open bullet.

Figure 7

Models of LAT-1 Signaling (A) Summary figure of structure-function correlation for two different activities of the LAT-1 receptor. Two models of receptor function incorporate these findings. (B and C) Homodimerization through the RBL domain (a) initiates the 7TM-dependent LAT-1 activity (b). (B) In the bidirectional signaling model, the LAT-1 ectodomain interacts in trans with a molecule on the adjacent membrane through which a reverse signal (c) is conveyed to the neighboring cell. (C) The bimodal forward signaling model places LAT-1 in a cis-interaction with a molecule on the same cell membrane. This model accommodates a coreceptor sensing the same stimulus as the LAT-1 ectodomain and transducing a signal (c) parallel to the 7TM-dependent signal of LAT-1.

Figure S1

Organization of the 7TM Receptor Superfamily and Protein Domain Composition in Selected aGPCR Members, Related to Figure 1. (A) 7TM receptors can be subgrouped according to the GRAFS classification (Schiöth and Fredriksson, 2005). aGPCR constitute the second largest 7TM receptor class with more than 30 members in mammalian species, which can be further classified in eight groups (Bjarnadóttir et al., 2004). Only latrophilin and CELSR/Flamingo aGPCR (bold) are conserved in invertebrate genomes (Nordström et al., 2009). (B) Representative members of the main aGPCR groups with positions of protein domains in the N-terminus drawn to scale. Note the distance similarity of the HRM-GPS-7TM motif region (boxed in gray) among different aGPCRs. B1/Secretin receptors (bottom) contain only a HRM but no GPS, whereas the PKD1 protein (top) possesses a similar juxtamembrane GPS-7TM design as aGPCRs. (C) aGPCR possess a GPS (a-c) in different domain contexts: cleavable GPS-HRM (a); cleavable GPS-(no HRM)-EGF (b); non-cleavable GPS-HRM (c). B1/secretin-like receptor for comparison (d).

Figure S2

Membrane Targeting of LAT-1::GFP Receptor Variants Is Not Impaired, Related to Figures 2 and 4. (A–D) Confocal images show comparable sections of the terminal bulb of the adult pharynx; anterior to the left. LAT-1::GFP variants colocalize with the membrane marker FM4-64. Inset shows higher magnification of the boxed cell. Dotted arrow indicates direction and length of plot axis in A’-D’. (A’–D’) Sectional line plots of signal intensity profiles of both channels underscores co-localization of LAT-1::GFP fusion proteins and FM4-64 intensity peaks at the membrane (M) and low signal intensities in the cytoplasm (C). (E) A signal generated by a soluble GFP does not overlap with the membrane marker FM4-64. Inset: higher magnification of the boxed cell. Dotted arrow indicates direction and length of plot axis in E’’. (E’) Sectional line plot of the signal intensity profile of both channels shows segregated intensity peaks for FM4-64 at the plasma membrane (M) and of lat-1p::GFP in the cytosol (C) indicating that both labels do not colocalize. Scale bars = 5 μm.

Figure S3

Quantitative Analysis of Membrane Targeting of LAT-1::GFP Receptor Variants, Related to Figures 2 and 4. (A) Confocal section through the head region of an adult worm expressing a soluble GFP chromophore under the identical lat-1 promoter and regulatory elements which control expression of all assayed LAT-1::GFP fusion proteins. Strong GFP expression can be seen in pharyngeal muscle (arrowheads) and the nervous system (arrows). Scale bar = 10 μm. (B) Intensity correlation analysis of LAT-1::GFP receptor variants/FM4-64 costains. All assayed fusion proteins show dependent (i.e., colocalized) staining with the membrane marker, which is statistically indistinguishable from the LAT-1::GFP control fusion. This indicates that molecular manipulations did not disrupt membrane targeting, whereas the soluble GFP control shows segregation (i.e., non-colocalization) from the FM4-64 signal. Numbers of analyzed individuals per transgenic strain in brackets. Bold horizontal lines in the box plot represent the medians, boxes the 25% and 75% quartiles, and whiskers the minimum and maximum values of the dataset.

Figure S4

Presence but Not Cleavage of the GPS Is Conserved in Most aGPCRs, Related to Figure 4. (A) Alignment of GPS with complete consensus sequence for autocatalytic cleavage. The His residue at position −2 and the S/T residues at position +1 relative to cleavage (arrowhead) are marked with arrows. The Cys residue mutated in GPR56 is in position 497 of the alignment. Residue numbering is according to the LAT-1A protein sequence. (B) Alignment of GPS that do not conform to the consensus sequence for autocatalytic cleavage. Note that several members of the CELSR/Fmi family in insects, which are orthologous to the cleavable Drosophila FMI, do not possess the consensus cleavage motif. Loss of cleavage activity has been experimentally confirmed for GPS of aGPCR shaded in gray. Species abbreviations: ag/Anopheles gambiae, am/Apis mellifera, ce/Caenorhabditis elegans, dm/Drosophila melanogaster, hs/Homo sapiens, mm/Mus musculus.