Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family

Abstract

The activity of the DAF-2 insulin-like receptor is required for Caenorhabditis elegans reproductive growth and normal adult life span. Informatic analysis identified 37 C. elegans genes predicted to encode insulin-like peptides. Many of these genes are divergent insulin superfamily members, and many are clustered, indicating recent diversification of the family. The ins genes are primarily expressed in neurons, including sensory neurons, a subset of which are required for reproductive development. Structural predictions and likely C-peptide cleavage sites typical of mammalian insulins suggest that ins-1 is most closely related to insulin. Overexpression of ins-1, or expression of human insulin under the control of ins-1 regulatory sequences, causes partially penetrant arrest at the dauer stage and enhances dauer arrest in weak daf-2 mutants, suggesting that INS-1 and human insulin antagonize DAF-2 insulin-like signaling. A deletion of the ins-1 coding region does not enhance or suppress dauer arrest, indicating a functional redundancy among the 37 ins genes. Of five other ins genes tested, the only other one bearing a predicted C peptide also antagonizes daf-2 signaling, whereas four ins genes without a C peptide do not, indicating functional diversity within the ins family.

Figure 1

Domain structure and genomic organization of the ins gene family. (A) All insulin superfamily genes encode at least a signal sequence, B and A domains. The signal sequence is removed during transit into the endoplasmic reticulum. (Pre) Signal sequence. Arrowheads indicate possible sites of proteolytic processing. (B) Domain organization of prepro- and mature insulin superfamily proteins. Canonical disulfide bonds are those contained in vertebrate insulin. Orange boxes indicate amino acid insertions into loops between the α-helices of the B and A chains. Black arrowheads indicate predicted processing sites for removal of the signal sequence, and red arrowheads indicate other predicted proteolytic processing sites. Predicted disulfide bonds and cysteine locations in blue indicate locations identical to disulfide bonds in vertebrate insulin. Disulfide bonds and cysteine locations in red indicate additional predicted disulfide bonds not contained in vertebrate insulin and cysteine locations that vary from the exact spacing in vertebrate insulin. We established the cDNA sequence of ins-1 through ins-31 and a transcript from ins-33 has been identified independently (M. Hristova and V. Ambros, pers. comm.). Because all of the tested genes are expressed, few, if any, of the predicted genes are pseudogenes.

Figure 2

Sequence diversity of ins genes. Sequence alignment of B and A domains. Cysteine residues are in red type and boxed. Hydrophobic residues important for helix formation are overlaid in green. The conserved glycine at B8 is overlaid in yellow. The nonpolar residues that replace the A16/A11 disulfide bond are overlaid in purple. An intron is inserted at the end of the B domain except where indicated by solid lines between the domains. The sizes (in residues) of cleaved or potentially cleavable C domains are indicated by numbers in parentheses between the B and A domain sequences. Actual or predicted dibasic residue cleavage sites for prohormone convertase are underlined. The ins genes defined previously are ins-2 through ins-7, ins-11, and ins-21 through ins-23 (Duret et al. 1998), ins-1 (Gregoire et al. 1998), and ins-18 (Kawano et al. 2000).

Figure 3

Genomic organization of the ins gene family on C. elegans chromosomes (Roman numerals). Many ins genes are organized in clusters of from two to seven genes of the same class (Fig. 1B). γ-type ins genes are shown in yellow, β-type genes in red, α-type genes in blue, and the gene encoding a triplicated INS protein in purple. The sizes of clusters of three or more genes are indicated. The only intervening genes within the clusters are three genes between ins-13 and ins-12 (indicated by //). Arrowheads indicate relative direction of transcription. The left end of each chromosome is at the top. The relative distances between the genes are in map units (scale bar, 10 map units).

Figure 4

Increased gene dosage of ins-1 and human insulin enhances daf-2(e1365) dauer formation and lengthens wild-type life span. (A) The percentage of dauers formed at 20°C by daf-2(e1365) animals carrying the indicated transgenes was scored 3 d after eggs were laid. These data are from Table 1. (B) ins-1 transgenic daf-2(e1365) animals were derived from founders injected with the indicated concentrations of ins-1 DNA. Dauer arrest was assayed as in A and the average for six independent lines at each ins-1 DNA concentration is presented. The percent dauer arrest ranges for the six lines were 0%–52% (2.5 ng/μL), 0%–80% (5 ng/μL), 6%–64% (10 ng/μL), and 5%–70% (20 ng/μL). (C) Mean adult life span at 26°C was 10.6 d for ins-1(2091);him-5 (▴, n = 46) and 9.8 d for him-5 (solid line, n = 46). (D) Mean adult life span of wild-type transgenic worms at 26°C was 13.0 d for the ins-1 (•, n = 48), 13.7 d for the P_ins-1∷human insulin (▪, n = 48), and 11.1 d for no (solid line, n = 50) transgene. (E) Diagram of the ins-1 genomic region. The open arrowhead indicates the point of fusion in P_ins-1∷GFP. The solid arrowheads indicate the points at which the human insulin cDNA is fused in P_ins-1∷hIns. The 1.3-kb ins-1(nr2091) deletion removes the second and third exons.

Figure 5

Spatial expression of representative ins genes. All animals are adults oriented with anterior to the left. (A) GFP expression under control of the ins-9 promoter in a single bilaterally symmetric amphid neuron process (arrow) and cell body (arrowhead). (B) GFP expression driven by the ins-22 promoter in multiple amphid cell bodies (arrowheads mark two) and fasciculated sensory processes (arrow). (C) ins-1∷GFP expression in amphid neurons (vertical arrow marks sensory processes) and pharyngeal neurons (diagonal arrow marks cell process). (D) GFP expression under control of the ins-4 promoter in the hypodermis (hyp; or epidermis) at the surface of the animal. Regions with reduced fluorescence overlie positions of the body wall muscles, in which the hypodermal layer is much thinner. The hypodermis is helically twisted because of the transformation marker rol-6. GFP expression in neurons is not visible in this focal plane.