Functional characterization of GNAS mutations found in patients with pseudohypoparathyroidism type Ic defines a new subgroup of pseudohypoparathyroidism affecting selectively Gsα-receptor interaction

Abstract

Pseudohypoparathyroidism type Ia (PHPIa) is caused by GNAS mutations leading to deficiency of the α-subunit of stimulatory G proteins (Gsα) that mediate signal transduction of G protein-coupled receptors via cAMP. PHP type Ic (PHPIc) and PHPIa share clinical features of Albright hereditary osteodystrophy (AHO); however, in vitro activity of solubilized Gsα protein is normal in PHPIc but reduced in PHPIa. We screened 32 patients classified as PHPIc for GNAS mutations and identified three mutations (p.E392K, p.E392X, p.L388R) in four unrelated families. These and one novel mutation associated with PHPIa (p.L388P) were introduced into a pcDNA3.1(-) expression vector encoding Gsα wild-type and expressed in a Gsα-null cell line (Gnas(E2-/E2-) ). To investigate receptor-mediated cAMP accumulation, we stimulated the endogenous expressed β(2) -adrenergic receptor, or the coexpressed PTH or TSH receptors, and measured the synthesized cAMP by RIA. The results were compared to receptor-independent cholera toxin-induced cAMP accumulation. Each of the mutants associated with PHPIc significantly reduced or completely disrupted receptor-mediated activation, but displayed normal receptor-independent activation. In contrast, PHPIa associated p.L388P disrupted both receptor-mediated activation and receptor-independent activation. We present a new subgroup of PHP that is caused by Gsα deficiency and selectively affects receptor coupling functions of Gsα.

Figure 1

Stimulation of PHPIc associated missense mutants Gsα-388R and Gsα-392K. Panel a) shows cAMP accumulation of both transfected missense mutants after receptor mediated stimulation of the endogenous expressed β₂-receptor due to Isoproterenol compared to receptor-independent stimulation due to CTX. The mutation p.L388R results in a loss of receptor-mediated stimulation despite normal receptor-independent activation compared to the wild-type. The Gsα-392K mutant demonstrates a diminished activity after receptor-mediated stimulation, although the receptor-independent stimulation is comparable to that of the Gsα-wild-type. % max: maximal response after stimulation of the Gsα-wild type with CTX. Panel b) Different concentrations of Isoproterenol (Iso) ranging from 10⁻⁸ to 10⁻⁴ M lead to a dose dependent stimulation of the β₂ receptor in the wild-type and the Gsα-392K mutant. The Gsα-388R mutant failed to induce intracellular cAMP synthesis. Panel c) Similar results are shown after cotransfection with the PTHR: incubation with various concentrations of human PTH ranging from 10⁻¹¹ to 10⁻⁷ M lead to a dose dependent response for the wild-type, and the Gsα-392K mutant, whereas the Gsα-388R-mutant does not show any response.

Figure 1

Stimulation of PHPIc associated missense mutants Gsα-388R and Gsα-392K. Panel a) shows cAMP accumulation of both transfected missense mutants after receptor mediated stimulation of the endogenous expressed β₂-receptor due to Isoproterenol compared to receptor-independent stimulation due to CTX. The mutation p.L388R results in a loss of receptor-mediated stimulation despite normal receptor-independent activation compared to the wild-type. The Gsα-392K mutant demonstrates a diminished activity after receptor-mediated stimulation, although the receptor-independent stimulation is comparable to that of the Gsα-wild-type. % max: maximal response after stimulation of the Gsα-wild type with CTX. Panel b) Different concentrations of Isoproterenol (Iso) ranging from 10⁻⁸ to 10⁻⁴ M lead to a dose dependent stimulation of the β₂ receptor in the wild-type and the Gsα-392K mutant. The Gsα-388R mutant failed to induce intracellular cAMP synthesis. Panel c) Similar results are shown after cotransfection with the PTHR: incubation with various concentrations of human PTH ranging from 10⁻¹¹ to 10⁻⁷ M lead to a dose dependent response for the wild-type, and the Gsα-392K mutant, whereas the Gsα-388R-mutant does not show any response.

Figure 1

Stimulation of PHPIc associated missense mutants Gsα-388R and Gsα-392K. Panel a) shows cAMP accumulation of both transfected missense mutants after receptor mediated stimulation of the endogenous expressed β₂-receptor due to Isoproterenol compared to receptor-independent stimulation due to CTX. The mutation p.L388R results in a loss of receptor-mediated stimulation despite normal receptor-independent activation compared to the wild-type. The Gsα-392K mutant demonstrates a diminished activity after receptor-mediated stimulation, although the receptor-independent stimulation is comparable to that of the Gsα-wild-type. % max: maximal response after stimulation of the Gsα-wild type with CTX. Panel b) Different concentrations of Isoproterenol (Iso) ranging from 10⁻⁸ to 10⁻⁴ M lead to a dose dependent stimulation of the β₂ receptor in the wild-type and the Gsα-392K mutant. The Gsα-388R mutant failed to induce intracellular cAMP synthesis. Panel c) Similar results are shown after cotransfection with the PTHR: incubation with various concentrations of human PTH ranging from 10⁻¹¹ to 10⁻⁷ M lead to a dose dependent response for the wild-type, and the Gsα-392K mutant, whereas the Gsα-388R-mutant does not show any response.

Figure 2

PHPIc and PHPIa associated Gsα-mutants can be distinguished by in vitro stimulation tests. These experiments demonstrate that the cell model may be appropriate to reflect differences in receptor-independent activation between PHPIc and PHPIa and between the effects of missense and nonsense mutations: Panel a) In contrast to Gsα-388R, the Gsα-388P mutant found in a patient with PHPIa leads to a clearly diminished CTX induced cAMP synthesis that was significantly different from the corresponding cAMP levels in cells expressing Gsα-388R (P<0.001). Panel b) After receptor-mediated stimulation the Gsα-392K mutant revealed a residual activity that was absent in the nonsense mutant Gsα-392X (P<0.001). Panel c) Comparison of Gsα-protein levels in non-transfected (n.t.) and transfected Gnas^E2−/E2− cells by immunoblot analysis. Gsα-wild-type (WT), Gsα-388R (388R), Gsα-388P (388P), Gsα-392K (392K), and Gsα-392X (392X). The protein levels of the mutants were similar to those of the wild-type.

Figure 2

PHPIc and PHPIa associated Gsα-mutants can be distinguished by in vitro stimulation tests. These experiments demonstrate that the cell model may be appropriate to reflect differences in receptor-independent activation between PHPIc and PHPIa and between the effects of missense and nonsense mutations: Panel a) In contrast to Gsα-388R, the Gsα-388P mutant found in a patient with PHPIa leads to a clearly diminished CTX induced cAMP synthesis that was significantly different from the corresponding cAMP levels in cells expressing Gsα-388R (P<0.001). Panel b) After receptor-mediated stimulation the Gsα-392K mutant revealed a residual activity that was absent in the nonsense mutant Gsα-392X (P<0.001). Panel c) Comparison of Gsα-protein levels in non-transfected (n.t.) and transfected Gnas^E2−/E2− cells by immunoblot analysis. Gsα-wild-type (WT), Gsα-388R (388R), Gsα-388P (388P), Gsα-392K (392K), and Gsα-392X (392X). The protein levels of the mutants were similar to those of the wild-type.

Figure 2

PHPIc and PHPIa associated Gsα-mutants can be distinguished by in vitro stimulation tests. These experiments demonstrate that the cell model may be appropriate to reflect differences in receptor-independent activation between PHPIc and PHPIa and between the effects of missense and nonsense mutations: Panel a) In contrast to Gsα-388R, the Gsα-388P mutant found in a patient with PHPIa leads to a clearly diminished CTX induced cAMP synthesis that was significantly different from the corresponding cAMP levels in cells expressing Gsα-388R (P<0.001). Panel b) After receptor-mediated stimulation the Gsα-392K mutant revealed a residual activity that was absent in the nonsense mutant Gsα-392X (P<0.001). Panel c) Comparison of Gsα-protein levels in non-transfected (n.t.) and transfected Gnas^E2−/E2− cells by immunoblot analysis. Gsα-wild-type (WT), Gsα-388R (388R), Gsα-388P (388P), Gsα-392K (392K), and Gsα-392X (392X). The protein levels of the mutants were similar to those of the wild-type.

Figure 3

Ribbon representation of Gsα in complex with GTP-γs. Left: The C-terminal helix (α5) is colored in light blue, the helix α4 and the β-strand β6 in green and the GTP-γs molecule is depicted as a ball and stick representation. C denotes the C-terminus. Right: Close-up of the C-terminal region. The side chain of L388 is depicted and the hydrogen bond between the amide group of L388 and the carbonyl group of Q384 is shown in red (dashed line).