ABSTRACT
Glucagon-like peptide 1 receptor (GLP-1R) controls diverse physiological functions in tissues including the pancreatic islets, brain, and heart. To understand the mechanisms that control glucagon-like peptide 1 (GLP-1) signaling better, we sought to identify proteins that interact with the GLP-1R using a membrane-based split ubiquitin yeast two-hybrid (MYTH) assay. A screen of a human fetal brain cDNA prey library with an unliganded human GLP-1R as bait in yeast revealed 38 novel interactor protein candidates. These interactions were confirmed in mammalian Chinese hamster ovarian cells by coimmunoprecipitation. Immunofluorescence was used to show subcellular colocalization of the interactors with GLP-1R. Cluster analysis revealed that the interactors were primarily associated with signal transduction, metabolism, and cell development. When coexpressed with the GLP-1R in Chinese hamster ovarian cells, 15 interactors significantly altered GLP-1-induced cAMP accumulation. Surprisingly, all 15 proteins inhibited GLP-1-activated cAMP. Given GLP-1's prominent role as an incretin, we then focused on 3 novel interactors, SLC15A4, APLP1, and AP2M1, because they are highly expressed and localized to the membrane in mouse insulinoma ß-cells. Small interfering RNA-mediated knockdown of each candidate gene significantly enhanced GLP-1-induced insulin secretion. In conclusion, we have generated a novel GLP-1R-protein interactome, identifying several interactors that suppress GLP-1R signaling. We suggest that the inhibition of these interactors may serve as a novel strategy to enhance GLP-1R activity.
Subject(s)
Protein Interaction Maps , Receptors, Glucagon/metabolism , Animals , CHO Cells , Cricetinae , Cricetulus , Cyclic AMP/metabolism , Glucagon-Like Peptide 1/pharmacology , Glucagon-Like Peptide-1 Receptor , HEK293 Cells , Humans , Insulin/metabolism , Insulin Secretion , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Mice , Protein Binding/drug effects , Protein Interaction Maps/drug effects , Reproducibility of Results , Two-Hybrid System Techniques , Ubiquitin/metabolismABSTRACT
The distal portion of the long arm of porcine chromosome 1 has been shown to harbour several quantitative trait loci affecting growth and reproductive traits in swine. In order to identify potential candidate genes that might underlie these effects, a comparative mapping analysis was undertaken to define the extent of orthologous segments of human chromosome 9. A microsatellite associated with heat shock protein (HSP) A5 was used to define the proximal boundary of the quantitative trait loci (QTL) region, which suggests the human orthologue of the gene(s) responsible for the observed effects lies between HSPA5 and the q arm telomere of human chromosome 9. Examination of this region revealed two candidate genes with known roles in production of hormones essential to growth and reproductive function. The steroidogenic factor 1 and Lhx3 LIM homeodomain transcription factor genes were mapped to 123 and 155 cM, respectively, of the Sus scrofa chromosome 1 (SSC1) linkage group, placing both genes within the confidence interval for the observed QTL. To further evaluate Lhx3, we examined the expression profile during porcine embryonic development. Low levels were detected at early embryonic stages, when development of the nervous system is proceeding. A transient increase in expression level is observed during the time of pituitary organogenesis and again at the time of differentiation of anterior pituitary cells, with relatively high levels of expression persisting in the adult pituitary gland. This ontology is consistent with Lhx3 being a candidate gene for the QTL.
Subject(s)
DNA-Binding Proteins/genetics , Heat-Shock Proteins , Homeodomain Proteins/genetics , Quantitative Trait, Heritable , Reproduction , Swine/genetics , Transcription Factors/genetics , Animals , Carrier Proteins/genetics , Chromosome Mapping/veterinary , DNA-Binding Proteins/physiology , Embryo, Mammalian/metabolism , Endoplasmic Reticulum Chaperone BiP , Fushi Tarazu Transcription Factors , Gene Expression , Homeodomain Proteins/physiology , LIM-Homeodomain Proteins , Molecular Chaperones/genetics , Physical Chromosome Mapping/veterinary , Receptors, Cytoplasmic and Nuclear , Sexual Maturation/genetics , Steroidogenic Factor 1 , Swine/embryology , Swine/growth & development , Transcription Factors/physiologyABSTRACT
The cause of posterior pituitary ectopia associated with anterior pituitary hormone deficiencies is unknown. We describe children with combined pituitary hormone deficiency (CPHD) or isolated GH deficiency. In all cases, magnetic resonance imaging examination revealed abnormal pituitary gland development featuring ectopic posterior lobe location and frequently hypoplastic anterior lobes. Embryonic development of the pituitary requires the coordinated expression of specific transcription factors. Mutations of the PIT-1 and PROP-1 transcription factors are responsible for CPHD in some patients with normally positioned posterior pituitaries. In mice, the Lhx3 LIM homeodomain transcription factor is required for both structural development and cellular differentiation of the pituitary gland. Thus, we hypothesized that mutations in one or both of the two human LHX3 isoforms are responsible for posterior pituitary ectopia associated with anterior pituitary hypopituitarism. Comprehensive molecular analysis of the LHX3 isoforms was performed to test this hypothesis. No loss of function mutations in the LHX3 gene were detected. In addition, analysis of PROP-1 did not reveal mutations that might cause this phenotype. These studies suggest that the abnormal processes leading to the development of CPHD or GH deficiency associated with posterior pituitary ectopia are not a result of aberrant LHX3 or PROP- 1 function, but may be caused by defects at other gene loci.
Subject(s)
Choristoma/genetics , Homeodomain Proteins/genetics , Hypopituitarism/genetics , Pituitary Diseases/genetics , Pituitary Gland/abnormalities , Pituitary Hormones/deficiency , Animals , Child , Child, Preschool , DNA-Binding Proteins/genetics , Female , Gene Deletion , Humans , Hypopituitarism/pathology , Infant , LIM-Homeodomain Proteins , Magnetic Resonance Imaging , Male , Mice , Phenotype , Pituitary Gland/pathology , Pituitary Gland, Anterior , Protein Isoforms/genetics , Transcription Factor Pit-1 , Transcription Factors/geneticsABSTRACT
The Lhx3 LIM homeodomain transcription factor is critical to pituitary organogenesis and motor neuron development. We determined the genomic structure and chromosomal localization of human LHX3. The gene contains seven coding exons and six introns that span 8.7 kilobases in length. The LHX3 gene codes for two functionally distinct isoforms that differ in their amino termini but share common LIM domains and a homeodomain. The functional domains of the LHX3 proteins are encoded by distinct exons. The alternate amino termini and LIM domains lie within individual exons, and the homeodomain is coded by two exons interrupted by a small intron. Human LHX3 maps to the subtelomeric region of chromosome 9 at band 9q34.3, within a region noted for chromosomal translocation and insertion events. Characterization of the genomic organization and chromosomal localization of LHX3 will enable molecular evaluation and genetic diagnoses of pituitary diseases and central nervous system developmental disorders in humans.
