Wu W I, Schwindinger W F, Aparicio L F, Levine M A
Division of Pediatric Endocrinology, Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, USA.
J Biol Chem. 2001 Jan 5;276(1):165-71. doi: 10.1074/jbc.M006032200.
G(s) is a heterotrimeric (alpha, beta, and gamma chains) G protein that couples heptahelical plasma membrane receptors to stimulation of adenylyl cyclase. Inactivation of one GNAS1 gene allele encoding the alpha chain of G(s) (G alpha(s)) causes pseudohypoparathyroidism type Ia. Affected subjects have resistance to parathyroid hormone (PTH) and other hormones that activate adenylyl cyclase plus somatic features termed Albright hereditary osteodystrophy. By contrast, subjects with pseudohypoparathyroidism type Ib have hormone resistance that is limited to PTH and lack Albright hereditary osteodystrophy. The molecular basis for pseudohypoparathyroidism type Ib is unknown. We analyzed the GNAS1 gene for mutations using polymerase chain reaction to amplify genomic DNA from three brothers with pseudohypoparathyroidism type Ib. We identified a novel heterozygous 3-base pair deletion causing loss of isoleucine 382 in the three affected boys and their clinically unaffected mother and maternal grandfather. This mutation was absent in other family members and 15 additional unrelated subjects with pseudohypoparathyroidism type Ib. To characterize the signaling properties of the mutant G alpha(s), we used site-directed mutagenesis to introduce the isoleucine 382 deletion into a wild type G alpha(s) cDNA, transfected HEK293 cells with either wild type or mutant G alpha(s) cDNA, plus cDNAs encoding heptahelical receptors for PTH, thyrotropic hormone, or luteinizing hormone, and we measured cAMP production in response to hormone stimulation. The mutant G alpha(s) protein was unable to interact with the receptor for PTH but showed normal coupling to the other coexpressed heptahelical receptors. These results provide evidence of selective uncoupling of the mutant G alpha(s) from PTH receptors and explain PTH-specific hormone resistance in these three brothers with pseudohypoparathyroidism type Ib. The absence of PTH resistance in the mother and maternal grandfather who carry the same mutation is consistent with current models of paternal imprinting of the GNAS1 gene.
G(s)是一种异源三聚体G蛋白（由α、β和γ链组成），它将七螺旋质膜受体与腺苷酸环化酶的激活偶联起来。编码G(s)α链（Gα(s)）的一个GNAS1基因等位基因失活会导致Ia型假性甲状旁腺功能减退。受影响的个体对甲状旁腺激素（PTH）和其他激活腺苷酸环化酶的激素有抵抗作用，并伴有称为奥尔布赖特遗传性骨营养不良的躯体特征。相比之下，Ib型假性甲状旁腺功能减退患者的激素抵抗仅限于PTH，且无奥尔布赖特遗传性骨营养不良。Ib型假性甲状旁腺功能减退的分子基础尚不清楚。我们使用聚合酶链反应分析了来自三名患有Ib型假性甲状旁腺功能减退的兄弟的基因组DNA中的GNAS1基因，以寻找突变。我们在三名受影响男孩及其临床未受影响的母亲和外祖父中鉴定出一个新的杂合3碱基对缺失，导致异亮氨酸382缺失。其他家庭成员以及另外15名患有Ib型假性甲状旁腺功能减退的无关受试者中不存在这种突变。为了表征突变型Gα(s)的信号特性，我们使用定点诱变将异亮氨酸382缺失引入野生型Gα(s) cDNA，用野生型或突变型Gα(s) cDNA以及编码PTH、促甲状腺激素或促黄体生成素的七螺旋受体的cDNA转染HEK293细胞，并测量激素刺激后cAMP的产生。突变型Gα(s)蛋白无法与PTH受体相互作用，但与其他共表达的七螺旋受体的偶联正常。这些结果提供了突变型Gα(s)与PTH受体选择性解偶联的证据，并解释了这三名患有Ib型假性甲状旁腺功能减退的兄弟中PTH特异性激素抵抗的原因。携带相同突变的母亲和外祖父没有PTH抵抗，这与当前关于GNAS1基因父系印记的模型一致。
