Bourne lab Home

Human G protein mutations

Pseudohypoparathyroidism, type I (PHP-I), is an inherited human disease caused by mutational inactivation of the alpha subunit of Gs (alpha-s), the stimulatory regulator of adenylyl cyclase. We discovered the alpha-s defect in PHP-I, and have recently elucidated two interesting and instructive mutations in patients with PHP-I.

Taroh Iiri studied effects of the alpha-s mutation found in rare patients with a combination of PHP-I and testotoxicosis, a disorder characterized by autonomous production of testosterone, producing precocious puberty in males. Substitution of a serine for a conserved alanine at position 366 in alpha-s caused this combination of rare disorders in two unrelated patients. The mutation constitutively activates alpha-s by accelerating release of bound GDP, which is then replaced by GTP, thus mimicking receptor activation; this effect accounts for the gain-of-function disorder, testotoxicosis. PHP-I in these patients is caused by a second effect of the mutation, thermolability. The protein denatures at body temperature (37¡) but not at testis temperature (33¡). The location of the alpha-s-A366S mutation in the 3D structure of G-alpha subunits suggests that a short loop of G-alpha, which touches the guanine ring of GDP, participates in the receptor-induced conformational change that results in GDP release.

Zvi Farfel, an alumnus of the lab now at Tel Aviv University, discovered the second mutation in a family with classical PHP-I (without testotoxicosis). The mutation substitutes histidine for a conserved arginine at position 231 of alpha-s, in the switch 2 region of G-alpha. Taroh found that the R231H mutation causes a subtle but functionally critical GTP binding defect which prevents activation of the G protein by receptors, AlF4-, and cholera toxin. The corresponding arginine is conserved in the alpha-2 helix of all Ga proteins. 3D structures of G-alpha subunits reveal that binding GTP causes this helix to move toward the guanine nucleotide and to twist about its axis to form a coordinated complex with residues in the alpha-3 helix and the preceding loop. In this complex the conserved arginine side chain (mutated in the family studied by Zvi) forms a salt bridge with a conserved glutamate in the alpha-3 helix, positioning alpha-2 precisely with respect to alpha-3; in addition, its guanidinium group stabilizes the main chain oxygen of the same glycine whose amide group interacts with the gamma-phosphate of GTP. The R231H phenotype suggests that the salt bridge serves as an intramolecular hasp to fasten together the alpha-2 and alpha-3 helices, allowing G-alpha to hold GTP tightly and maintain the active conformation more effectively.

This laboratory has for many years studied human diseases involving trimeric G-proteins. In earlier efforts, the laboratory described the molecular mechanism of cholera, identifying the G-alpha residue modified by cholera toxin. This modification constitutively activates alpha-s by slowing hydrolysis of GTP, the reaction that turns off G proteins. We also discovered constitutively activating alpha-s mutations in pituitary tumors of patients with acromegaly (excess of Growth Hormone); these mutations also slow the G-alpha turnoff reaction. The molecular pathogenesis of acromegaly is the same as that of cholera: most of the pituitary mutations substitute other amino acids for the same arginine residue that is modified by cholera toxin. This finding suggested that the arginine side chain plays a catalytic role in the GTPase reaction; 3D structures of G-alpha subunits have confirmed this prediction.

Last updated 10/01

G protein alpha subunit in its GTP-bound form, highlighting amino acids changed by point mutations that cause human endocrine diseases. Mutational replacements of red residues impair GTP hydrolysis; these sites are mutated in growth hormone secreting tumors of the pituitary. Replacement of either cyan residue produces an inactive G protein alpha subunit, causing pseudohypoparathyroidism. Bound nucleotide is light green.