Naturally occurring mutations of GPCRs

A number of diseases have already been attributed to mutational defects of GPCRs and with the techniques now available to isolate and sequence genes many more are likely to be found. Genetic polymorphisms are frequently occurring genetic variants within

Figure 230 Polymorphisms of human p2-adrenergic receptors. Circles indicate positions of most common polymorphisms and their functional significance. Reprinted from Trends in Pharmacological Science, 20, Buscher, R., Herrmann, V. and Insel, P. A., Human adrenoceptor polymorphisms: evolving recognition of clinical importance, 94-99. Copyright (1999), with permission from Elsevier.

Figure 230 Polymorphisms of human p2-adrenergic receptors. Circles indicate positions of most common polymorphisms and their functional significance. Reprinted from Trends in Pharmacological Science, 20, Buscher, R., Herrmann, V. and Insel, P. A., Human adrenoceptor polymorphisms: evolving recognition of clinical importance, 94-99. Copyright (1999), with permission from Elsevier.

a population and, in this respect, p2-adrenergic receptors are known to be highly polymorphic (Figure 230) and at least four polymorphisms are known in which an individual amino acid is different to that of the wild-type receptor. Further in vitro and in vivo studies are likely to extend the range of known mutations/genetic polymorphisms and provide a better insight concerning those which might predispose an individual to the onset of a disease, alter the clinical course of a disease or the response to clinical treatment.

GPCR mutations can be grouped according to whether they:

• Do not affect receptor function: for example, Val34Met has not yet been associated with changes in p2-adrenergic receptor function.

• Cause a loss of function: they may be autosomal recessive so that the lack of receptor function can be compensated in heterozygotes by the normal gene product. Hence, they are only apparent in homozygotes. Alternatively, they may be autosomal dominant and cannot be compensated. For example, Thr164Ile for the P2-adrenergic receptor leads to several functional effects, including lower binding affinities of the messenger and to a deficient coupling of the receptors to the adenylate cyclase system (Figure 230).

• Cause constitutive activation of the receptors: the role of CAMs in human disease was first demonstrated in 1993 for the thyrotropin (TSH) receptor in hyperfunctioning thyroid adenoma. Several dozen mutations affecting the TM or extracellular domains of this GPCR constitutively activate the cAMP signalling pathway. This in turn activates thyroid hormone secretion, resulting in hyperfunctioning thyroid adenoma (somatic mutations) or familial hyperthyroidism (germinal mutations). This example was followed by many others (Table 26). There is growing evidence that somatic GPCR-activating mutations are also involved in cell growth, and probably also in the development

Table 26 Hereditary diseases linked to naturally occurring mutations in different GPCRs Reprinted from Proceedings of the National Academy of Sciences, 97, Parnot, C., Bardin, S., Miserey-Lenkei, S., Guedin, D., Corvol, P. and Clauser, E., Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay, 7615-7620. Copyright (2000), with permission from Elsevier.

Table 26 Hereditary diseases linked to naturally occurring mutations in different GPCRs Reprinted from Proceedings of the National Academy of Sciences, 97, Parnot, C., Bardin, S., Miserey-Lenkei, S., Guedin, D., Corvol, P. and Clauser, E., Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay, 7615-7620. Copyright (2000), with permission from Elsevier.

Receptor

Mutations

Phenotype

Disease

TSH receptor

50 mutations: S281N/IT, R310C,

Hyperthyroidism

Somatic: thyroid

A339"367, S425L, G431S, M453T,

toxic adenoma

I486F/M, I586T, V597L, S505R/

Germinal: familial

N, V509A, L512E/R, I568M/T,

hyperthyroidism

A613-621 AD6!9 D6WG

T620S, A623I/S/V, L629F,

i630l, f631l/c/i, t632i/a,

D633A/Y/E/H, P639A, N650Y,

A658-661 V656F F666S N670S

C672Y, N674D, L677V

LH receptor

14 mutations: M398T, L457R,

Male precocious

Somatic: Leydig

i542l, d564g, a568v, m571i,

puberty

tumor and

A572V, I574L, I575L, T577I,

precocious puberty

d578h/g/y, c581r

Germinal: sporadic

and familial

precocious puberty

FSH receptor

1 mutation: D567G

Male fertility after

hypophysectomy

Rhodopsin

4 mutations: G90D, E113Q,

Blindness

Germinal: stationary

and Opsins

A292E, K296N

night blindness,

retinitis pigmentosa

PTH receptor

3 mutations: H223R, T*10P,

Short-limb

Germinal: Jansen

I458R

dwarfism, skeletal

chondrodysplasia

deformities,

hypercalcemia

and low PTH

Ca2+ sensing

23 mutations: K47N, P55L,

Hypocalcemia and

Germinal: autosomal

receptor

R68C, N118K, F128L/A,

hypercalciuria

dominant

t151m, n178d, e191k, y218s,

hypocalcemia

P221S/L, P227L, E228Q,

q245r, f612s, p747l, l 773r,

F788C, V817I, A835T, A895-1075

of cancer. In this respect, several orphan GPCRs, such as the mas oncogene, have been identified by their tumorigenic properties.

• Affect receptor downregulation: for example, Gln27Glu is responsible for a decreased downregulation of the p2-adrenergic receptor while Arg16Gly leads to enhanced downregulation of this receptor (Figure 230). This latter mutation occurs more frequently in patients with nocturnal asthma. Clinical studies also unveil that, for African- Caribbeans, the frequency of this mutation is significantly higher in those who are essentially hypertensive than in those which have a normal blood pressure.

Variation in splicing is another important mechanism leading to physiological diversity among GPCRs. Many GPCR genes contain multiple exons. Normally, the introns are removed at the level of processing of pre-mRNAs in the cell nucleus. Nevertheless, GPCR variants may be obtained due to alternative splicing, exon skipping and intron retention (Figure 231). There are now over 30 GPCRs with identified splice variants:

• The largest number of splice variants is at the C-terminus of the receptors, but some receptors have more than one site for variation in splicing.

I e4 I genomic DNA

transcription

~el i—gt ^ag—I e2 ]—It ^g n ei_ J—GT AG—j_e£j—

Splicing

Alternative splicing I el I eg I e4 I I et I e3 I e4 I

Ex on skipping I el I ea I e3 I e~

.1

| e2 | 64 |

| e3 | e4 |

e1 ¡ e2 I la | e3 | e4 | | ei | 11 | eg | e3 | g4~

translation receptor variants

Figure 231 The process by which splice variants can be produced from a hypothetical gene (e = exons, i = introns). Reprinted from Trends in Pharmacological Science, 20, Kilpatrick, G. J., Dautzenberg, F. M., Martin, G. R. and Eglen, R. M., 7TM receptors: the splicing on the cake, 294-301. Copyright (1999), with permission from Elsevier.

• The extent of splice variants depends to some extent on the complexity of gene structure (i.e. the amount of introns) and the nature of the variation might be species-specific.

• Few splice variants affect the messenger-binding domain of the receptors, but splice variants can have profound effects on the signalling pathway (e.g. adenylate cyclase stimulation versus inhibition for two variants of the thromboxane A2 receptor) as well as on the coupling efficiency (especially when the variation affects intracellular domains of the receptor).

Splice variants of GPCRs are often dismissed as the consequence of leaky transcription and, hence, physiologically irrelevant. However, there are several reports linking splice variants with disease. The D3 dopamine receptor is reported to be associated with schizophrenia, and abnormal processing of the CCK-B receptor has been associated with gallstones and obesity.

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