Four Contiguous Amino Acid Substitutions, Identified in Patients with Laron Syndrome, Differently Affect the Binding Affinity and Intracellular Trafficking of the Growth Hormone Receptor¹

Abstract

We have analyzed the GH receptor (GHR) gene in four individuals with Laron syndrome, and a missense mutation was identified for each patient in the extracellular domain of the GHR (D152H, I153T, Q154P, and V155G). The D152H mutation was previously reported. We have reproduced the three novel mutations in the GHR complementary DNA and analyzed their consequences in human 293 transfected cells. In cells expressing the I153T and V155G mutants, binding of [125I]human GH at the cell surface was very low, whereas binding to total membrane fractions was much less affected, suggesting impaired cell surface expression. Binding assays with cells expressing the Q154P mutant revealed severe defects both at the cell surface and in total particulate membrane fractions. Immunofluorescence experiments confirmed that cell surface expression of the three mutants was altered, and colocalization studies suggested that most of the mutant receptors are retained in the endoplasmic reticulum. Endoglycosidase H resistance tests also indicated that the majority of I153T and V155G GHRs are trapped in the endoplasmic reticulum. Thus, mutations on contiguous amino acids of the GHR result in various defects. The I153T, Q154P, and V155G mutations mainly affect intracellular trafficking and binding affinity of the receptor, whereas the D152H mutation affects receptor expression, dimerization, and signaling.

Studies with a Growth Hormone Antagonist and Dual-fluorescent Confocal Microscopy Demonstrate that the Full-length Human Growth Hormone Receptor, but Not the Truncated Isoform, Is Very Rapidly Internalized Independent of Jak2-Stat5 Signaling

“…Labeling was present both at the cell surface and in intracellular compartments with a juxtanuclear accumulation in the Golgi apparatus. Such distribution has been described previously in COS-7 cells (26) and 293 cells (27) transfected with the GHR. In cells expressing the GHR1-317-EGFP, the fluorescence resulting from the antibody was superimposed on that from the GFP, so that even in the absence of Triton X-100, some intracellular labeling appeared because of the GFP.…”

A Recurrent Mutation in Growth Hormone Receptor (GHR) Gene Underlying Laron-type Dwarfism in a Pakistani Family

“…Another missense variant in the same codon (c.509A>G; p.(Asp170Gly)) has been reported in a Taiwanese family [ 34 ]. We concluded that the mutation underlies the pathogenesis since it segregates with the malformation in the family and has already been reported in two LS families of Asian origin [ 29 ] and another family from Pakistan [ 35 ]. Furthermore, the online bioinformatics tools predict this variant as pathogenic (Appendix A: Supplemental Table 3).…”

Serum Growth Hormone and Insulin-Like Growth Factor-1 Concentrations in Japanese Black Cattle with Growth Retardation.

“…There are many possible reasons for this phenomenon. In human patients with Laron syndrome [13], a noncontiguous loss of exons 3, 5 and 6 [5] or a series of mutations in the extracellular domain of the GH receptor [19,21] were revealed. Miniature Bos Indicas cattle, which had characteristics similar to human Laron type dwarfism, had reduced expressions of mRNAs of GH receptor 1A in the liver [13].…”