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G protein Mutations Causing Disease LiYang Slide 2 GPCRs are known for their physiological functions. Induction of these diverse biological functions results from the activation of a collection of heterotrimeric G- proteins, which consist of subunits and closely associated - subunits. Slide 3 G-proteins, which consist of subunits and closely associated - subunits. The subunit is responsible for GTP and GDP binding and for GTP hydrolysis, whereas the and subunits are associated in a tightly linked - complex. Slide 4 Following a brief overview of G protein coupled signal transduction, we review the growing body of evidence that mutations in genes encoding GPCRs and G proteins are an important cause of human disease. Slide 5 Sorting of the diseases according the mechanism Inactive or absent Gs (subunit) Constitutively active Gs (subunit) Temperature-sensitive Gs Constitutively active Gi 2 Slide 6 gsp mutations of Gs G protein 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 subunit, causing pseudohypoparathyroidism. Bound nucleotide is light green. Slide 7 1.Inactive or absent Gs (a subunit) Pseudohypoparathyroidism-- type I (PHP-I), is an inherited human disease caused by mutational inactivation of the subunit of Gstype I (PHP-I), Multiple endocrine abnormalities in cAMP regulated organs Occurs when bad gene inherited from mother Pseudopseudohypoparathyroidism--clinically less severe syndrome, same as mutation in one Gs Occurs when bad gene inherited from father Both conditions--as protein levels about half of normal Slide 8 Pseudohypoparathyroidism Patients with this disease, which manifests in a short stature, round face, one or more foreshortened metacarpal or metatarsal bones (particularly the fourth metacarpal), obesity, and subcutaneous calcifications. Albright's hereditary osteodystrophy Slide 9 Pseudohypoparathyroidism One inactive copy of Gs Resistant to PTH , TSH , ACTH (and others) Complex tissue-specific genetic imprinting: syndrome worse if inherit bad copy from mom than from dad Slide 10 2. Constitutively active Gs (a subunit) Tumors Mutations in s --block GTPase activity, cause constitutive activity as candidate oncogene (termed gsp) Activating mutations found in 40% of growth hormone secreting pituitary adenomas; found in other endocrine tumors including pituitary, thyroid McCune Albright Syndrome Somatic mutation in s in early embryonic development Patients mosaic for constitutively active Gs (as) Slide 11 McCune-Albright Syndrome. Somatic mutation of Gs alpha early in development Effects of activating MSH and gonadotrophin receptors evident Slide 12 3.Temperature-sensitive s --"Testotoxicosis" Testotoxicosis the gain-of-function disorder Symptoms: Males have general features of precocious puberty and hypoparathyroidism with a mutant as that is inactive at 37C and constitutively active at testicular temperature Slide 13 Both patients were found to contain a single amino acid substitution in one of the Gisoforms. The alternation in amino acid sequence caused two effects on the mutant G protein. precocious puberty--indicating premature testicular activation (normally testosterone production is stimulated by LH, a GPCR coupled to cAMP formation) hypoparathyroidism--impaired responses to PTH , TSH causing PTH and thyroid abnormalities Slide 14 At temperatures below normal body temperature, the mutant G protein remained in the active state, even in the absence of a bound ligand. In contrast, at normal body temperatures, the mutant G protein was inactive, both in the presence and absence of bound ligand, the testes, which are housed outside of the bodys core, have a lower temperature than the bodys visceral organs (33 versus 37 ). Slide 15 precocious puberty Normally, the endocrine cells of the testes initiate testosterone production at the time of puberty in response to the pituitary hormone LH, which begins to be produced at that time. The circulating LH binds to LH receptors on the surface of the testicular cells, inducing the synthesis of cAMP and subsequent production of the male sex hormone, the testicular cells of the patients bearing the G protein mutation were stimulated to synthesize cAMP in the absence of the LH ligand, leading to premature synthesis of testosterone and precocious puberty. Slide 16 Hypoparathyroidism In contrast, the mutation in this same G subunit in the cells of the parathyroid glands,which function at a temperature of 37 , caused the G protein to remain inactive. As a result, the cells of the parathyroid gland could not respond to stimuli that would normally cause them to secrete parathyroid hormone, leading to the condition of hypoparathyroidism. The fact that most of the bodily organs functioned in a normal manner in these patients suggests that this particular G isoform is not essential in the activities of most other cells. Slide 17 Testotoxicosis Conclusion: Syndrome due to a temperature-sensitive Gs alpha Protein inactive at 37 but constitutively active at the lower temperature of the testes at32C 37 33 normal -s-A366S mutation Gs cAMP Produced in cyc- cells normal mutation 37 33 Western Blot for Gs alpha Slide 18 4 Constitutively active Gi 2 Tumors Mutations in ai2 --block GTPase activity, cause constitutive activity i2 candidate oncogene (termed gip) Activating mutations found in >30% of adrenal, ovarian tumors Slide 19 compare ovarian tumor Slide 20 THE END