Variable and tissue-specific hormone resistance in heterotrimeric Gs protein alpha-subunit (Gsalpha) knockout mice is due to tissue-specific imprinting of the gsalpha gene

Abstract

Albright hereditary osteodystrophy (AHO), an autosomal dominant disorder characterized by short stature, obesity, and skeletal defects, is associated with heterozygous inactivating mutations of GNAS1, the gene encoding the heterotrimeric G protein alpha-subunit (Gsalpha) that couples multiple receptors to the stimulation of adenylyl cyclase. It has remained unclear why only some AHO patients present with multihormone resistance and why AHO patients demonstrate resistance to some hormones [e.g., parathyroid hormone (PTH)] but not to others (e.g., vasopressin), even though all activate adenylyl cyclase. We generated mice with a null allele of the mouse homolog Gnas. Homozygous Gs deficiency is embryonically lethal. Heterozygotes with maternal (m-/+) and paternal (+/p-) inheritance of the Gnas null allele have distinct phenotypes, suggesting that Gnas is an imprinted gene. PTH resistance is present in m-/+, but not +/p-, mice. Gsalpha expression in the renal cortex (the site of PTH action) is markedly reduced in m-/+ but not in +/p- mice, demonstrating that the Gnas paternal allele is imprinted in this tissue. Gnas is also imprinted in brown and white adipose tissue. The maximal physiological response to vasopressin (urinary concentrating ability) is normal in both m-/+ and +/p- mice and Gnas is not imprinted in the renal inner medulla (the site of vasopressin action). Tissue-specific imprinting of Gnas is likely the mechanism for variable and tissue-specific hormone resistance in these mice and a similar mechanism might explain the variable phenotype in AHO.

Figure 1

Generation of Gnas knockout (GsKO) mice. (A) Schematic diagram of the wild type Gnas allele (Top) with exons 1–3 depicted as black boxes. The targeting vector (Middle) was generated by insertion of the GK-Neo cassette into a BamHI (B) site within exon 2 in the same orientation as Gnas (arrow), followed by digestion with SmaI and SstI (the SstI site is contributed by the vector). The recombinant GsKO allele is shown below. The position of the 5′ probe, a NotI-SmaI fragment, is shown above. After digestion with XmnI (X), this probe is predicted to hybridize to an 11-kb genomic fragment in the wild type allele and a 9-kb genomic fragment in the GsKO allele. The position of the 3′ probe used to confirm correct insertion of the targeting vector at the 3′ end is shown above. Southern hybridization using this probe is not shown. (B) Southern hybridization of F1 progeny derived from mating of a GsKO chimera with a CD1 mouse. Genomic DNA was digested with XmnI and hybridized with the 5′ probe. The genotype of individual animals is indicated above each lane.

Figure 2

M−/+ and +/p− neonates have distinct physical characterisitics. Shown are (a) +/p−, (b) m−/+ neonates above and (c, d) normal littermates below. Note lack of milk in stomach of +/p− pup.

Figure 3

bPTH(1–34)-stimulated cAMP production in isolated renal proximal tubules. Proximal tubule suspensions were incubated with the indicated concentrations of bPTH(1–34) in the presence of 0.5 mM IBMX. Maximal cAMP response was achieved in normal mice at a concentration of 3.45 × 10⁻⁸. Data are the mean ± SEM of 3 animals for +/p− and m−/+ mice and 6 animals for +/+ mice (except at the highest bPTH concentration in which the data are from 5 animals). Asterisks indicate that the response was significantly reduced in m−/+ mice at all concentrations of agonist.

Figure 4

G_sα expression in renal cortex and inner medulla and white and brown adipose tissue. In A–C the results of paired littermates (normal +/+, mutant +/−, or −/+) are shown. (A) Immunoblots of renal cortical membranes (above, CTX, 8 μg protein/lane) and renal inner medulla membranes (below, IM, 5 μg protein/lane) incubated with RM antibody followed by ¹²⁵I-protein A. (B) Immunoblots of membranes prepared from adipocytes isolated from gonadal fat pads (3 μg protein/lane) incubated with RM antibody. (C) Northern hybridization of total RNA isolated from intrascapular brown adipose tissue (4 μg/lane) with a ³²P-labeled G_sα rat cDNA probe.

Figure 5

In situ hybridization of renal cortex from adult mice. Bright field microscopic images of renal cortex from (A) +/p−, (B) m−/+, and (C) +/+ mice after hybridization with antisense Gnas riboprobe and (D) +/+ mouse after hybridization with sense Gnas riboprobe. Glomeruli are indicated with arrows. (×400.)